Inhibition of Rac1 attenuated propofol-induced neurotoxicity in the hippocampal dentate gyrus in developing mice

Inhibition of Rac1 attenuated propofol-induced neurotoxicity in the hippocampal dentate gyrus in developing mice | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Inhibition of Rac1 attenuated propofol-induced neurotoxicity in the hippocampal dentate gyrus in developing mice Yuan Li, Haifeng Duan, Qi Wang, Yi Lin, Zhoujing Yang, Zhiru Wang, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6288169/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 25 Jun, 2025 Read the published version in Neurochemical Research → Version 1 posted 10 You are reading this latest preprint version Abstract Accumulating evidence from animals has shown that multiple exposures to general anesthetics during brain development may cause extensive neuronal apoptosis and long-term memory impairments. However, the underlying mechanism is still poorly understood. In the present study, C57BL/6 mice were administered propofol at postnatal days 19–21. We found that propofol exposure significantly increased neuronal apoptosis in the mouse hippocampal dentate gyrus, as evidenced by cleaved caspase-3 (c-c3) immunofluorescence. Additionally, propofol exposure increased the activation and expression of Ras-related C3 botulinum toxin substrate 1 (Rac1) in the dentate gyrus, as shown by western blotting. Modulating Rac1, either through inhibition or overexpression, was found to mitigate or exacerbate propofol-induced neuronal apoptosis, respectively. We further demonstrated that propofol decreased the expression of the antiapoptotic protein phosphorylated serine/threonine kinase Akt via Rac1. The results of the open field and morris water maze tests further revealed that silencing Rac1 alleviated propofol-induced cognitive dysfunction. Our findings demonstrated that propofol exposure induced neuronal apoptosis and long-term cognitive dysfunction in the mouse hippocampal dentate gyrus by regulating Rac1. Propofol Dentate gyrus Neuronal apoptosis Cognitive dysfunction Rac1 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Background Millions of children require general anesthesia worldwide because of the need for surgery or medical procedures every year[1]. However, numerous animal studies have demonstrated that early exposure to general anesthetics in the developing brain may cause neuronal apoptosis and neurocognitive impairment[2-5]. Some retrospective human studies also reported that repeated exposure to general anesthesia in children is associated with the development of cognitive and behavioral deficits later in life[6-8]. Consequently, the United States Food and Drug Administration (FDA) has issued a warning emphasizing the potential neurotoxic effects of general anesthetics on the developing brains of children under the age of three and on fetuses when administered to pregnant women (www.fda.gov/Drugs/DrugSafety/ucm532356.htm). Propofol is one of the most commonly used intravenous anesthetic agents in children because of its rapid onset and rapid recovery following discontinuation of administration[9]. Like other general anesthetics, early exposure to propofol also causes neuronal apoptosis in the developing brain, raising serious concerns about the safety of propofol anesthesia in children[10, 11]. Nevertheless, the molecular and cellular mechanisms underlying the effects of early propofol exposure on brain development remain poorly understood. Therefore, investigating the signaling pathways involved in propofol-induced neuronal apoptosis is imperative. Ras-related C3 botulinum toxin substrate 1 (Rac1), a small Rho guanosine triphosphate phosphohydrolases (GTPases), is recognized for its responsiveness to environmental stress in cells or organisms. It detects external signals from receptors on the membrane and triggers downstream signaling pathways to perform its physiological functions[12]. Rac1 is vital for regulating several cellular processes, including cytoskeletal structure, oxidative product generation, and gene expression[13]. Specifically, Rac1 plays a crucial role in brain functions such as neuronal migration, synaptic plasticity, and memory formation by regulating actin dynamics in neurons[14]. Irregular Rac1 expression and activity have been noted in various neurological disorders[15]. Recently, the role of Rac1 in neuroapoptosis has attracted increasing attention. Research has indicated that the inhibition of Rac1 activity ameliorates neuronal apoptosis and cognitive deficits caused by cerebral ischemia‒reperfusion in the hippocampal CA1 region of rats[16]. Additionally, neurotrophic factors alleviate propofol-induced neuronal apoptosis in the hippocampus, a process contingent upon Rac1 expression[17]. However, whether Rac1 is involved in propofol-induced neurotoxicity remains unknown. Therefore, the aim of the present work was to determine whether Rac1 participates in propofol-induced neurotoxicity. In our study, repeated exposure to propofol induced widespread apoptosis in the developing dentate gyrus (DG), accompanied by increased expression of Rac1. Modulating Rac1, either through inhibition or overexpression, can mitigate or exacerbate propofol-induced neuronal apoptosis, respectively. Moreover, we found that Rac1 silencing mitigated propofol-induced long-term cognitive dysfunction. These results indicate that Rac1 might be a promising therapeutic target for the clinical treatment of propofol-induced neurotoxicity. Materials and methods Experimental animals C57BL/6 mice were used. The procedures adhered to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Ethical Committee for Animal Research of Fudan University (No. 201802130S). All the mice were reared in temperature- and humidity-controlled rooms with a 12-h light/dark cycle. Efforts were made to reduce the number of animals used to minimize the number required for statistical accuracy and minimize suffering. Both male and female mice were used in this study. Anesthesia The mice were randomly divided into a control group and a propofol group. In the propofol group, the mice were injected intraperitoneally with 150 mg/kg propofol from P19 to P21 once a day for three consecutive days. After the injection, they were placed on a constant temperature blanket at 37°C for 6 h to prevent hypothermia. On the third day after the last injection, the animals were euthanized 6 h after complete recovery, and samples were collected for western blot and immunofluorescence analysis. Injection of agonists/inhibitors The specific agonist CN04-A (848 µmol/L, 1 µl/side, Cytoskeleton, USA) and the inhibitor NSC23766 (125 µmol/L, 1 µl/side, Tocris, UK) for Rac1 activity were dissolved in ultrapure water and freshly prepared on the day of the experiment. P19 mice were anesthetized via intraperitoneal injection of pentobarbital and secured in a stereotaxic frame. After shaving and disinfecting, the scalp was incised, and the subcutaneous tissue was separated to fully expose the skull. The anterior fontanelle, posterior fontanelle, coronal suture, and sagittal suture were adequately revealed. A cranial drill was subsequently used at the DG site (with the anterior fontanelle as the reference point, ML = ± 1.5 mm, AP = -2 mm, and DV = -2.5 mm) to gently create an opening in the skull for needle insertion. Glass electrode filaments were pulled, and their tips were observed under a microscope. A diameter of 50 µm was left at the tip, prefilled with mineral oil to eliminate air, and connected to a microsyringe (1 ml) via hot melt adhesive. The microsyringe was then slowly injected with 1 µl/side of the solvent into the dentate gyrus of both sides of the mouse, taking over 5 min for the injection. Afterward, the microsyringe was left in place for 5 min, followed by slow withdrawal to prevent reflux. Postoperatively, the area was disinfected, sutured, and marked for subsequent experimental injections. Injection of Adeno-associated virus Short hairpin RNA (shRNA) targeting Rac1 was constructed by Obio Technology in Shanghai, China. This shRNA was inserted into an adeno-associated virus (AAV) vector carrying green fluorescent protein (GFP). This shRNA is designed to specifically target mouse neurons, and it can specifically downregulate or upregulate the expression of Rac1. The same vector backbone was used to construct a negative control. For virus brain stereotaxic microinjections in P3 mice, the mice were anesthetized through intraperitoneal injection of pentobarbital and secured in a stereotaxic frame. Then, with a microsyringe (1 ml) connected to a glass electrode with a tip diameter of 50 µm, 1 µl of the virus was slowly injected into the DG on both sides of the mice (with the anterior fontanelle as the reference point, ML = ± 1.0 mm, AP = -1.5 mm, DV = -2 mm) over a period exceeding 5 min. Finally, the microsyringe was left in place for 5 min, followed by slow withdrawal to prevent reflux. Immunohistochemistry, image acquisition and analysis Six hours after saline or anesthesia injection, all pups were deeply anesthetized with 0.7% sodium pentobarbital and perfused with 0.9% saline, followed by 4% paraformaldehyde (PFA) in PBS. The brains were dissected and postfixed in 4% PFA/PBS for 4–6 h at 4°C and equilibrated in 30% sucrose. Coronal sections (30 µm) containing the DG were cut using a Leica CM1950 cryostat (Wetzlar, Germany), and every 5th section was immunostained. The sections were incubated in a blocking solution containing 5% bovine serum albumin (BSA) and 0.5% Triton X-100 for 2 h at 37°C. A primary rabbit monoclonal antibody against cleaved caspase-3 (c-c3, 9661 L, 1:400; Cell Signaling Technology, Beverly, MA, USA) was applied overnight in 0.3% BSA at 4°C. After being washed three times in PBS, the sections were incubated with an Alexa Fluor-conjugated (488 or 568 nm) secondary antibody at 1:500 (Thermo Fisher Scientific, Waltham, MA, USA) for 2 h at 37°C. Nuclei were labeled with 4′,6-diamidino-2-phenylindole (DAPI) (Thermo Fisher Scientific) for 30 min at 37°C. The stained slices were scanned via the TissueFAXS Plus system. The number of cleaved caspase-3 (c-c3)-positive cells was quantified. Following the completion of all the image scans, the image data were processed and analyzed via image analysis software (ImageJ, version 1.48 V, USA). Costaining of c-c3 and DAPI nuclear staining was used to identify apoptotic neurons. All the data were subjected to blind analysis via Image-Pro Plus software (Media-Cybernetics, Rockville, MD, USA) under experimental conditions. The cells were considered c-c3 positive if they were larger than the threshold size and displayed detectable cell contours. Approximately 40 c-c3-positive cells were imaged and analyzed per mouse. To analyze the proportion of c-c3-positive cells, all images were thresholded before counting, and colocalization was defined as overlap between thresholded regions. For all the experiments, brain tissue sections from the control and anesthetized mice were coprocessed, including those from the immunostaining, imaging, and analysis steps. Each experiment involved the analysis of 10 image frames from each mouse. Arterial blood gas measurements Arterial blood gas was measured in mic at 3 h or 6 h following propofol anesthesia. Arterial blood was obtained via transcardiac aspiration of the left ventricle via a heparinized 32-gauge hypodermic needle. The pH, partial pressure of carbon dioxide (PaCO 2 ), PaO 2 , SaO 2 , and HCO 3− concentrations were measured immediately after arterial blood collection via a portable clinical analyzer (ABL800FLEX, Radiometer, Copenhagen, Denmark). Pull-down assay To prepare the tissue lysate, the tissue was homogenized in prechilled 1× lysis buffer (#20–168, Millipore) and placed on ice for 30 min. Afterward, it was centrifuged at 4000 × g for 15 min at 4°C, and the supernatant was collected. The sample concentration was determined via the BCA protein assay method. Pak1 is an effector protein that can bind to active Rac1. To detect this interaction, 1 mg of total protein was incubated with 10 µl of PAK-1 PBD agarose beads for 1 h. The agarose beads were subsequently washed three times, resuspended in 40 µl of 2× reducing sample buffer and boiled for 5 min. The bound proteins were separated via 12.5% SDS‒PAGE and detected by immunoblotting with a Rac1 antibody (#05‒389, Millipore) to determine the level of GTP-bound Rac1. Western blotting The mice were deeply anesthetized via i.p. injection of sodium pentobarbital, followed by decapitation, after which the regions encompassing the bilateral DGs were dissected at designated time points and preserved in liquid nitrogen. Next, total proteins were extracted from the DGs by homogenizing the tissues in ice-cold RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China) supplemented with 0.1 mM phenylmethylsulfonyl fluoride-protease inhibitors and phosphatase inhibitors, lysing them on ice for 30 min, and centrifuging them at 4°C for 10 min. Samples were heated at 95°C for 10 min in loading buffer before equal amounts of protein were loaded onto a 10% sodium dodecyl sulfate–polyacrylamide gel for electrophoretic separation (SDS‒PAGE) and then transferred onto polyvinylidene difluoride membranes at 4°C. After being blocked with 5% nonfat milk for 2 h at room temperature, the membranes were incubated overnight at 4°C with a primary antibody (1:1000 dilution) or β-actin (1:500 dilution, Boster #BM3873, Wuhan, China). The membranes were washed with PBS buffer and incubated with horseradish peroxidase-conjugated secondary anti-rabbit IgG (1:2000 dilution; Abcam #ab6721, Cambridge, MA) for 2 h at room temperature, and the bands were visualized via enhanced chemiluminescence (ECL, Thermo Fisher China, Shanghai, China). The relative levels of the target protein normalized to the level of β-actin were determined via densitometry analysis via ImageJ software (National Institutes of Health, Bethesda, MD, USA). Morris water maze Behavioral assessments were conducted in a quiet, light-regulated environment. Prior to initiating the water maze experiments, the mice underwent a three-day habituation period to familiarize them with the testing room and experimenters. Both groups of mice were introduced into a circular plastic pool with a diameter of 120 cm, filled with water that had been dyed black. The water temperature was consistently maintained at 23 ± 1°C. Visual cues were strategically placed around the perimeter of the pool to assist the mice in locating the submerged platform. For each trial, the mice were gently introduced into the pool from one of four preassigned entry points and given 60 s to find a platform submerged 2 cm below the water surface. The platform location was randomized among the four quadrants but remained consistent for each individual mouse during the acquisition phase. If a mouse failed to locate the platform within the allotted time, it was guided to the platform. Once on the platform, the mice remained there for 30 s. At the conclusion of each trial, the animals were removed from the pool, dried with a towel, and returned to their home cages. The acquisition phase spanned five consecutive days, with one session per day consisting of four trials, each separated by a 30 min interval. The trials were recorded on video, and the swimming trajectories were analyzed via water maze software. The parameters included the time taken to locate the platform and the swimming speed calculated for each trial and averaged over daily sessions. A probe test was conducted 24 h after the training phase concluded. During this test, the platform was removed, and the animals were allowed to swim freely for 60 seconds. The time spent in the target zone and the number of taget crossings were calculated to evaluate the long-term memory of the platform's previous location. Open Field Test The open field test is a widely used method for assessing both anxiety-related behaviors and locomotor activity. The test was conducted via a Tru Scan (USA) open field apparatus consisting of a 1-meter diameter square arena equipped with infrared sensors to monitor animal movements in both horizontal and vertical dimensions. The mice were individually placed in the open field for a 30 min observation period. The data collected included the total distance traveled and the mean speed, the distance occurring within the central zone, and the time spent in the central area. The behavioral metrics for each mouse were processed and analyzed via TRU SCAN2.1 software. Statistical analysis Statistical analyses were performed via GraphPad Prism 6 (GraphPad Software, La Jolla, CA, United States). One-way analysis of variance (ANOVA, comparing three or more conditions) followed by Dunnett’s test (all conditions compared to the control) and two-way ANOVA (for comparing two independent variables) were used to assess statistical significance, followed by Bonferroni’s or Tukey’s multiple comparisons test, as specified in the figure legends. The data are shown as the means ± SEMs. P < 0.05 was considered statistically significant, and all the conditions significantly different from the controls were reported. Results Propofol induces cell apoptosis in the dentate gyrus of the hippocampus in developing mice To investigate the impact of propofol exposure on neuronal apoptosis in the DG, C57BL/6 mice were treated with propofol from postnatal days 19 to 21 over a span of three consecutive days. Initially, we assessed the survival rates of mice subjected to various dosages of propofol. Our findings indicated that mice could tolerate doses of 100 mg/kg and 150 mg/kg propofol. However, a marked decrease in the survival rate was observed at a dosage of 200 mg/kg propofol (Fig. 1 A). Consequently, a dosage of 150 mg/kg was selected for the subsequent experiments. Furthermore, compared with that of the control group, 150 mg/kg propofol exposure significantly increased the number of apoptotic cells in the DG, as measured by quantitative analysis of c-c3 (Fig. 1 B, C). To evaluate whether propofol anesthesia affects the physiological state of mice, arterial blood gas analysis was performed at 3 h and 6 h after propofol anesthesia. Compared with the control group, propofol anesthesia had no effect on the pH, partial pressure of carbon dioxide (PaCO 2 ), partial pressure of oxygen (PaO 2 ), bicarbonate (HCO 3 ), or oxygen saturation (SaO 2 ), suggesting that a dosage of 150 mg/kg propofol did not alter the physiological state of the mice (supplementary Table 1). Propofol exposure leads to cognitive dysfunction in adulthood In our previous study, exposure of P21 mice to sevoflurane, a widely used general anesthetic, did not affect spatial or memory ability. However, whether repeated exposure to propofol at P21 affects cognitive impairment remains unknown. We initially explored the impact of propofol exposure on long-term cognitive function in developing mice. C57BL/6 mice were treated with propofol at P19-P21, and learning and memory performance in adulthood (P49–P55) was subsequently examined via the open field test and Morris water maze test. Compared with the control group, the open field test revealed no statistically significant differences in total distance traveled or average movement speed (Fig. 2 A‒C). However, compared with the control treatment, propofol treatment decreased the time spent and the frequency of visits to the central area (Fig. 2 A, D, E). Similarly, in the Morris water maze test, propofol treatment significantly decreased the number of times crossing the target zone and shortened the time spent in the target zone (Fig. 2 H, I). There was no significant difference in swimming speed and escape latency between the propofol group and the control group (Fig. 2 F, G). These results showed that propofol exposure causes long-term cognitive dysfunction in developing mice. Rac1 is activated and upregulated in the dentate gyrus of the hippocampus upon propofol exposure Rac1 acts as a molecular switch, and upon binding to GTP, it forms an activated Rac1-GTP complex and affects downstream Rac1 signaling pathways[ 18 ]. The activation of Rac1 is crucial for brain development. To determine whether propofol exposure enhances Rac1 activation and expression, we analyzed the expression of Rac1-GTP and Rac1 in the hippocampal dentate gyrus by western blotting. Our results revealed that propofol treatment dramatically increased the expression of Rac1-GTP and Rac1 in the hippocampal dentate gyrus (Fig. 3 A-C), indicating that Rac1 is activated and upregulated upon propofol exposure. Inhibition of Rac1 activation attenuated propofol-induced neuroapoptosis in the DG To identify whether the activation of Rac1 in the DG contributes to propofol-induced neuroapoptosis, the Rac1 agonist CN04-A or the inhibitor NSC23766 was injected into the bilateral DG of P19 mice before propofol exposure. As shown in Fig. 4 A-B, compared with that in the control group, the expression of Rac1-GTP in the mice treated with CN04-A alone was significantly greater. In mice treated with propofol, CN04-A also significantly increased Rac1-GTP expression. In contrast, the Rac1 inhibitor NSC23766 significantly reduced Rac1-GTP expression in both the control and propofol-treated groups, indicating excellent activation or inhibition of Rac1 (Fig. 4 C-D). Thereafter, c-c3 immunofluorescence was performed to examine the effects of Rac1 activation or inhibition on propofol-induced neuroapoptosis in the DG. As shown in Fig. 4 E-G, compared with propofol alone, CN04-A significantly increased propofol-induced apoptosis. Conversely, treatment with NSC23766 markedly reduced propofol-induced apoptosis. Collectively, these results suggest that Rac1 activation plays a role in the neuroapoptosis triggered by propofol exposure. Knockdown of Rac1 mitigated propofol-induced neuroapoptosis in the DG To further examine the role of Rac1 expression in propofol-induced neuroapoptosis in the DG, we injected adeno-associated virus (AAV) carrying small-molecule shRNA-Rac1 for overexpression (ovRac1) and silencing (shRac1), along with a Rac1 overexpression empty AAV vector (C-ovRac1) and a silencing empty AAV vector (C-shRac1), into the DG region of P3 mice. When the mice reached P21, we conducted western blot analysis to assess Rac1 protein expression levels in the hippocampal DG region. The mice were randomly divided into two groups: the control group (Ctrl) and the propofol-treated group (P). Compared with the respective empty vector controls for each group, Rac1 overexpression led to a significant increase in Rac1 expression in both the Ctrl and P groups (Fig. 5 . A-B). Compared with the respective empty vector controls for each group, Rac1 silencing resulted in a significant decrease in Rac1 expression in both the Ctrl and P groups (Fig. 5 . C-D), with statistically significant differences. These findings indicate the successful establishment of Rac1 overexpression and silencing interference models. Moreover, our results revealed that Rac1 overexpression increased the number of c-c3-positive neurons in the DG region. Compared with those in the propofol plus empty viral vector group, the number of c-c3-positive neurons was greater in the propofol plus Rac1 overexpression group. Similarly, knocking down Rac1 expression relieved propofol-induced c-c3-positive neurons (Fig. 5 E-G). Taken together, these findings indicate that Rac1 is involved in propofol-induced cell apoptosis in the hippocampal dentate gyrus. Rac1 silencing mitigated propofol-induced long-term cognitive dysfunction Moreover, we examined the potential involvement of Rac1 in long-term cognitive dysfunction. The mice were randomly divided into a control group (Ctrl group), a propofol group (P group), a C-shRac1 group and a shRac1 group. AAV carrying the small molecule shRNA-Rac1 for silencing (shRac1) or the silencing empty AAV vector (C-shRac1) was administered into the DG region of P3 mice in the shRac1 group and the C-shRac1 group, respectively. The mice in the P, shRac1 and C-shRac1 groups were exposed to propofol from P19 to P21. Behavioral assessments were conducted when the mice in all four groups reached adulthood (postnatal days 49–55). As shown in Fig. 6 , compared with the C-shRac1 group, propofol treatment did not affect the total distance traveled or average movement speed in the open field experiments after the expression of Rac1 was knocked down (Fig. 6 A-C). However, the distance traveled and the time spent in the central area were ameliorated after the expression of Rac1 was knocked down (Fig. 6 D, E). In the Morris water maze test, there were no statistically significant differences in swimming speed or escape latency between the shRac1 and C-shRac1 groups (Fig. 6 F, G). However, compared with those in the P group and the C-shRac1 group, the number of times crossing the target zone in the shRac1 group was significantly greater (Fig. 6 H), and the time in the target zone was significantly greater (Fig. 6 I). Taken together, these results suggest that Rac1 silencing could mitigate propofol-induced long-term cognitive dysfunction. Propofol treatment decreased Akt expression in DG region cells via Rac1 Previous studies have shown that Akt is crucial in the regulation of cell apoptosis, whereas Rac1 exerts a regulatory influence on apoptosis by modulating Akt activation[ 19 , 20 ]. To determine whether propofol affects the expression of Akt proteins through Rac1 in the DG region, we first assessed the expression of p-Akt/Akt following propofol treatment. Our results showed that propofol exposure significantly decreased p-Akt expression (Fig. 7 A, B). Therefore, we explored the effects of Rac1 overexpression on the expression of Akt following propofol treatment. Compared with that in the control group, the expression of p-Akt significantly decreased in the Rac1-overexpressing group (OvRac1 group) and the propofol plus Rac1-overexpressing group (propofol + OvRac1 group) (Fig. 7 C-E). We further examined the effects of Rac1 knockdown on the expression of Akt. We found that knocking down the expression of Rac1 did not affect the expression of p-Akt. However, knocking down the expression of Rac1 significantly increased the expression of p-Akt after propofol treatment (Fig. 7 F-H; P < 0.01). Taken together, these results indicate that propofol decreased Akt expression in DG region cells via Rac1. Discussion Over the past several decades, numerous animal studies have demonstrated that early exposure to nearly all general anesthetics can induce widespread neuroapopsis in the neonatal brain and adversely affect long-term brain development[ 21 , 22 ]. Although various mechanisms have been proposed to be involved in the development of neuroapoptosis and cognitive dysfunction following anesthesia, the underlying mechanism is still elusive. In the present study, we observed that early exposure to propofol resulted in extensive cellular apoptosis within the hippocampal dentate gyrus of mice and revealed that Rac1 plays a vital role in propofol-induced cell apoptosis and, subsequently, long-term cognitive dysfunction. Since our earlier research indicated that anesthesia-induced neuroapoptosis in the DG of mice was postponed, reaching its peak at postnatal day 21 instead of P7[ 23 ], the present study exposed mice to propofol from postnatal days 19 to 21. The mice were exposed to propofol at their most vulnerable time to maximize the influence of DG granule cell apoptosis on their long-term behavior. In various studies focused on neuroapoptosis and long-term behavioral disorders caused by anesthesia in the developing brain, rodents were anesthetized at P4–P8[ 6 , 24 – 26 ]. This is very different from our study. The discrepancy may arise from the fact that different brain areas have distinct windows of susceptibility to neurotoxicity induced by anesthesia. Notably, the CA1 region of the hippocampus showed maximum vulnerability to anesthesia-induced neuroapoptosis approximately one week after birth. The CA1 region is also associated with learning and memory[ 27 ]. This could explain why P4–P8 mice are regularly used for evaluating neurotoxicity under anesthesia. Rac1 belongs to the family of classical Rho guanosine triphosphate phosphohydrolases (GTPases), which are recognized for their involvement in various cellular processes, including cytoskeletal organization, the regulation of gene expression, and cell migration[ 28 ]. However, few studies have investigated the regulatory function of Rac1 in propofol-induced neuroapoptosis in the developing brain. Interestingly, we found that Rac1 was upregulated and activated in the dentate gyrus in P21 mice. To elucidate the role of Rac1 activation in the neuroapoptosis induced by propofol, Rac1 agonists and inhibitors were administered. Immunofluorescence analysis of c-c3 expression in the DG revealed that Rac1 activation significantly potentiated propofol-induced apoptosis, whereas inhibition of Rac1 activity substantially mitigated this apoptotic effect. These findings suggest that propofol might modulate apoptosis in the DG region through the regulation of Rac1 activity. In addition, small molecule shRNA-Rac1-overexpressing and small molecule-silenced adeno-associated viruses were utilized to determine the direct effect of Rac1 expression on propofol-induced apoptosis in the DG. Under physiological conditions, the overexpression of Rac1 resulted in increased apoptosis, whereas the silencing of Rac1 did not significantly affect apoptosis in the DG region. These findings suggest a correlation between elevated Rac1 expression and cell apoptosis in the DG. Following treatment with propofol, Rac1 overexpression further augmented propofol-induced apoptosis in DG region cells. Notably, silencing Rac1 led to a significant reduction in propofol-induced apoptosis in the DG. Collectively, these findings indicate that propofol modulates cell apoptosis in the DG via a Rac1-dependent mechanism. As demonstrated in previous reports, Rac1 exerts a regulatory influence on apoptosis by modulating Akt activation[ 29 ]. Rac1 is located primarily in the cytoplasm of cells and plays a crucial role in the regulation of various cellular processes. When involved in cell apoptosis, Rac1 is activated and translocated to the inner surface of the plasma membrane, where it recruits Akt to the membrane. This recruitment allows Akt to undergo phosphorylation, thereby engaging in downstream signaling pathways related to cell growth, apoptosis, and other cellular events[ 30 , 31 ]. In the present work, we found that Rac1 overexpression decreased p-Akt expression regardless of propofol exposure. In addition, knocking down the expression of Rac1 significantly increased the expression of p-Akt after propofol treatment. These results indicate that propofol might decrease Akt expression in DG region cells through Rac1 activation. However, the precise mechanism underlying the interaction between Rac1 and Akt under propofol exposure remains to be elucidated. Our study demonstrated that repeated propofol exposure from P19 to P21 caused long-term cognitive dysfunction in developing mice, as measured by the open field test and Morris water maze test. These findings were slightly different from those of our previous study, which revealed that sevoflurane exposure at P21 had no effect on spatial learning or memory ability in the Morris water maze test[ 32 ]. While many studies have indicated that exposing newborn mice to general anesthetics negatively impacts long-term spatial learning and memory, some studies have reported no effect or even an improvement in performance in the Morris water maze test. Newborn rats exposed to 2.6% sevoflurane anesthesia for 2 hours on postnatal days 7, 14, and 21 showed no changes in escape latency or platform crossing frequency in the Morris water maze test during adolescence (P31) or adulthood (P91)[ 33 ]. Neonatal mice (P4–P6) exposed to 1.8% sevoflurane for 6 hours exhibited superior performance in the training phase of the Morris water maze test[ 34 ]. The inconsistency between studies might result from the various anesthetics administered to the mice, the time they were exposed, or the age of the subjects. Moreover, our study demonstrated that Rac1 not only participates in regulating propofol-induced apoptosis but also in regulating cognitive function impairment. Rac1 silencing mitigated propofol-induced cognitive dysfunction in the open-field test and Morris water maze test. Our findings align with those of previous studies, which have shown that inhibiting Rac1 activity significantly mitigates the hippocampal cell damage and neurocognitive impairments induced by sevoflurane in rats. These results indicate that Rac1 might be a promising therapeutic target for propofol neurotoxicity. Our study acknowledges certain limitations. Specifically, we have shown that propofol exposure leads to a reduction in Akt expression in cells within the DG region through the regulation of Rac1. However, it remains to be definitively established whether the Rac1‒Akt pathway is directly involved in propofol-induced neuroapoptosis. Additionally, we described the neuroapoptotic effect of propofol exposure on P21 mice, and whether the loss of neurons at P21 extends to cognitive impairment in adulthood needs further confirmation. Conclusions In conclusion, our study demonstrated that repeated propofol exposure during the development period of mice induced widespread neuroapoptosis in the DG, accompanied by the activation and upregulation of Rac1. Inhibiting the activation or overexpression of Rac1 attenuated propofol-induced neuroapoptosis. Furthermore, Rac1 silencing mitigated propofol-induced long-term cognitive dysfunction. The current work provides evidence that Rac1 could be a promising therapeutic target for the clinical treatment of neuroapoptosis and cognitive dysfunction induced by propofol. Declarations Funding This work was supported by the National Natural Science Foundation of China (82271292, 81730031 to Yingwei Wang), the Foundation of Shanghai Municipal Science and Technology Medical Innovation Research Project (23Y21900600 to Yingwei Wang), the Foundation of Shanghai Municipal Key Clinical Specialty (shslczdzk06901 to Yingwei Wang), the National Natural Science Foundation of China (82371285 to Daojie Xu), the Foundation of Shanghai Municipal Science and Technology Commission (22ZR1409600 to Daojie Xu), and the National Natural Science Foundation of China (82201421 to Qi Wang, 82101272 to Kai Wei). Competing interests The authors declare no competing interests. Authors' contributions Daojie Xu, Zhiru Wang, Yingwei Wang and Kai Wei contributed to conception and design of the study; Qi Wang, Yuan Li, Haifeng Duan, Yi Lin, Zhoujing Yang contributed to acquisition and analysis of data; Yuan Li, Daojie Xu, Yingwei Wang and Kai Wei contributed to draft the work or substantively revise it. All authors read and approved the final manuscript. Data Availability All data generated or analysed during this study are included in this published article. Ethics approval The study was approved in accordance with the Ethical Committee for Animal Research of Fudan University Acknowledgement The authors thank the entire staff of the Department of Anesthesiology, Huashan Hospital, Fudan University for critical discussions of this manuscript. References Walkden GJ, Gill H, Davies NM, Peters AE, Wright I, Pickering AE. Early Childhood General Anesthesia and Neurodevelopmental Outcomes in the Avon Longitudinal Study of Parents and Children Birth Cohort. Anesthesiology. 2020;133(5):1007-20. Meng C, Yao X-q, Chang R-j, Wang S-l, Wang X, Ma D-q, et al. Exogenous GM1 Ganglioside Attenuates Ketamine-Induced Neurocognitive Impairment in the Developing Rat Brain. Anesthesia & Analgesia. 2020;130(2):505-17. Zhang Y, Wu Z, Li X, Wan Y, Zhang Y, Zhao P. Maternal sevoflurane exposure affects differentiation of hippocampal neural stem cells by regulating miR-410-3p and ATN1. Stem Cell Res Ther. 2020;11(1). Dai J, Li X, Wang C, Gu S, Dai L, Zhang J, et al. Repeated neonatal sevoflurane induced neurocognitive impairment through NF-κB-mediated pyroptosis. J Neuroinflammation. 2021;18(1). Noguchi KK, Johnson SA, Dissen GA, Martin LD, Manzella FM, Schenning KJ, et al. Isoflurane exposure for three hours triggers apoptotic cell death in neonatal macaque brain. Br J Anaesth. 2017;119(3):524-31. Lin EP, Lee J-R, Lee CS, Deng M, Loepke AW. Do anesthetics harm the developing human brain? An integrative analysis of animal and human studies. Neurotoxicology and Teratology. 2017;60:117-28. Andropoulos DB. Effect of Anesthesia on the Developing Brain: Infant and Fetus. Fetal Diagn Ther. 2018;43(1):1-11. Sun LS, Li G, Miller TL, Salorio C, Byrne MW, Bellinger DC, et al. Association Between a Single General Anesthesia Exposure Before Age 36 Months and Neurocognitive Outcomes in Later Childhood. JAMA. 2016;315(21):2312-20. Alexander S, Kairalla JA, Gupta S, Hibbitts E, Weisman H, Anghelescu D, et al. Impact of Propofol Exposure on Neurocognitive Outcomes in Children With High-Risk B ALL: A Children's Oncology Group Study. J Clin Oncol. 2024;42(22):2671-9. ML. P, Y. H, IR. N, HH. P, JC. D, DM. R, et al. Propofol neurotoxicity is mediated by p75 neurotrophin receptor activation. Anesthesiology. 2012 Feb;116(2):352-61. Milanovic D, Pesic V, Loncarevic-Vasiljkovic N, Avramovic V, Tesic V, Jevtovic-Todorovic V, et al. Neonatal Propofol Anesthesia Changes Expression of Synaptic Plasticity Proteins and Increases Stereotypic and Anxyolitic Behavior in Adult Rats. Neurotox Res. 2017;32(2):247-63. Margiotta A, Bucci C. Coordination between Rac1 and Rab Proteins: Functional Implications in Health and Disease. Cells. 2019;8(5). Shi Y, Bollam Saumya R, White Shannon M, Laughlin Sean Z, Graham Garrett T, Wadhwa M, et al. Rac1-Mediated DNA Damage and Inflammation Promote Nf2 Tumorigenesis but Also Limit Cell-Cycle Progression. Dev Cell. 2016;39(4):452-65. Barbosa S, Greville-Heygate S, Bonnet M, Godwin A, Fagotto-Kaufmann C, Kajava AV, et al. Opposite Modulation of RAC1 by Mutations in TRIO Is Associated with Distinct, Domain-Specific Neurodevelopmental Disorders. The American Journal of Human Genetics. 2020;106(3):338-55. Wang X, Liu D, Wei F, Li Y, Wang X, Li L, et al. Stress-Sensitive Protein Rac1 and Its Involvement in Neurodevelopmental Disorders. Neural Plast. 2020;2020:1-11. Cookson MR, Raz L, Zhang Q-G, Zhou C-f, Han D, Gulati P, et al. Role of Rac1 GTPase in NADPH Oxidase Activation and Cognitive Impairment Following Cerebral Ischemia in the Rat. PLoS ONE. 2010;5(9). XUZE LI, ZHIFANG ZHAO, LINING HUANG, RONGTIAN KANG, XUEFANG LIU, DONG Z. The anti-apoptotic effect of nerve growth factor on propofol-induced neurotoxicity in hippocampal neurons is Rac1 dependent. Pharmazie. 2018;73:706-10. Haga RB, Ridley AJ. Rho GTPases: Regulation and roles in cancer cell biology. Small GTPases. 2016;7(4):207-21. Cheng K-C, Chen Y-H, Wu C-L, Lee W-P, Cheung CHA, Chiang H-C. Rac1 and Akt Exhibit Distinct Roles in Mediating Aβ-Induced Memory Damage and Learning Impairment. Mol Neurobiol. 2021;58(10):5224-38. Gong H-Y, Zheng F, Zhang C, Chen X-Y, Liu J-J, Yue X-Q. Propofol protects hippocampal neurons from apoptosis in ischemic brain injury by increasing GLT-1 expression and inhibiting the activation of NMDAR via the JNK/Akt signaling pathway. Int J Mol Med. 2016;38(3):943-50. Vutskits L, Xie Z. Lasting impact of general anaesthesia on the brain: mechanisms and relevance. Nature Reviews Neuroscience. 2016;17(11):705-17. Li X, Wei K, Hu R, Zhang B, Li L, Wan L, et al. Upregulation of Cdh1 Attenuates Isoflurane-Induced Neuronal Apoptosis and Long-Term Cognitive Impairments in Developing Rats. Frontiers Cellular Neuroscience. 2017;11:368. Hofacer RD, Deng M, Ward CG, Joseph B, Hughes EA, Jiang C, et al. Cell age-specific vulnerability of neurons to anesthetic toxicity. Ann Neurol. 2013;73(6):695-704. Gill H, Pickering AE. The effects of xenon on sevoflurane anesthesia-induced acidosis and brain cell apoptosis in immature rats. Paediatr Anaesth. 2021;31(3):372-4. Li H, Dai CL, Gu JH, Peng S, Li J, Yu Q, et al. Intranasal Administration of Insulin Reduces Chronic Behavioral Abnormality and Neuronal Apoptosis Induced by General Anesthesia in Neonatal Mice. Front Neurosci. 2019;13:706. Xu C, Niu JJ, Zhou JF, Wei YS. MicroRNA-96 is responsible for sevoflurane-induced cognitive dysfunction in neonatal rats via inhibiting IGF1R. Brain Res Bull. 2019;144:140-8. Deng M, Hofacer RD, Jiang C, Joseph B, Hughes EA, Jia B, et al. Brain regional vulnerability to anaesthesia-induced neuroapoptosis shifts with age at exposure and extends into adulthood for some regions. Br J Anaesth. 2014;113(3):443-51. Bailly C, Degand C, Laine W, Sauzeau V, Kluza J. Implication of Rac1 GTPase in molecular and cellular mitochondrial functions. Life Sci. 2024;342. Hu F, Li N, Li Z, Zhang C, Yue Y, Liu Q, et al. Electrical pulse stimulation induces GLUT4 translocation in a Rac‐Akt‐dependent manner in C2C12 myotubes. FEBS Lett. 2018;592(4):644-54. Vaidya RJ, Ray RM, Johnson LR. MEK1 restores migration of polyamine-depleted cells by retention and activation of Rac1 in the cytoplasm. American Journal of Physiology-Cell Physiology. 2005;288(2):C350-C9. Liu B, Xu Y, Zhang L, Yang X, Chen L, Liu Y, et al. Hypermethylation of DLG3 Promoter Upregulates RAC1 and Activates the PI3K/AKT Signaling Pathway to Promote Breast Cancer Progression. Evid Based Complement Alternat Med. 2021;2021:1-11. Wei K, Liu Y, Yang X, Liu J, Li Y, Deng M, et al. Bumetanide attenuates sevoflurane‐induced neuroapoptosis in the developing dentate gyrus and impaired behavior in the contextual fear discrimination learning test. Brain and Behavior. 2022;12(11). Liang X, Zhang Y, Zhang C, Tang C, Wang Y, Ren J, et al. Effect of repeated neonatal sevoflurane exposure on the learning, memory and synaptic plasticity at juvenile and adult age. Am J Transl Res. 2017;9(11):4974-83. Chen C, Shen FY, Zhao X, Zhou T, Xu DJ, Wang ZR, et al. Low-dose sevoflurane promotes hippocampal neurogenesis and facilitates the development of dentate gyrus-dependent learning in neonatal rats. ASN Neurology. 2015;7(2). Additional Declarations No competing interests reported. Supplementary Files Supplementarymaterial.docx Cite Share Download PDF Status: Published Journal Publication published 25 Jun, 2025 Read the published version in Neurochemical Research → Version 1 posted Editorial decision: Revision requested 28 Apr, 2025 Reviews received at journal 27 Apr, 2025 Reviewers agreed at journal 13 Apr, 2025 Reviewers agreed at journal 11 Apr, 2025 Reviews received at journal 08 Apr, 2025 Reviewers agreed at journal 27 Mar, 2025 Reviewers invited by journal 26 Mar, 2025 Editor assigned by journal 24 Mar, 2025 Submission checks completed at journal 24 Mar, 2025 First submitted to journal 23 Mar, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6288169","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":439933107,"identity":"c0ae7197-a2e1-407d-850e-06d586170ddc","order_by":0,"name":"Yuan Li","email":"","orcid":"","institution":"Huashan Hospital, Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Yuan","middleName":"","lastName":"Li","suffix":""},{"id":439933108,"identity":"cf04981c-80cf-49b6-bcfa-162cf5040730","order_by":1,"name":"Haifeng Duan","email":"","orcid":"","institution":"Huashan Hospital, Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Haifeng","middleName":"","lastName":"Duan","suffix":""},{"id":439933110,"identity":"091dc8a8-4f0a-4eb3-ac97-d5f3906a332a","order_by":2,"name":"Qi Wang","email":"","orcid":"","institution":"Huashan Hospital, Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Qi","middleName":"","lastName":"Wang","suffix":""},{"id":439933111,"identity":"ff62c6fb-42d5-4754-8988-f7753d826990","order_by":3,"name":"Yi Lin","email":"","orcid":"","institution":"Huashan Hospital, Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Yi","middleName":"","lastName":"Lin","suffix":""},{"id":439933112,"identity":"21f4d941-bccf-4636-bfe7-e1a28c3334ea","order_by":4,"name":"Zhoujing Yang","email":"","orcid":"","institution":"Huashan Hospital, Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Zhoujing","middleName":"","lastName":"Yang","suffix":""},{"id":439933114,"identity":"88f8a11e-9a05-4a7c-9cc8-5ecd489bd2b7","order_by":5,"name":"Zhiru Wang","email":"","orcid":"","institution":"East China Normal University","correspondingAuthor":false,"prefix":"","firstName":"Zhiru","middleName":"","lastName":"Wang","suffix":""},{"id":439933115,"identity":"9022406f-46ae-4e88-9b18-c7c84de9db9d","order_by":6,"name":"Yingwei Wang","email":"","orcid":"","institution":"Huashan Hospital, Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Yingwei","middleName":"","lastName":"Wang","suffix":""},{"id":439933116,"identity":"d1f116bd-8f20-4feb-be4d-96d01c7ae2f4","order_by":7,"name":"Kai Wei","email":"","orcid":"","institution":"Huashan Hospital, Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Kai","middleName":"","lastName":"Wei","suffix":""},{"id":439933117,"identity":"970bc278-6135-4c60-93a0-b4f58fe2c959","order_by":8,"name":"Daojie Xu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7klEQVRIiWNgGAWjYHACAwYGHgkGfuYDDBJg/gFitUi2JZCkBUQeI1aLwY3kjR8+yFjIGR/jMbzxcweDHN+NBMbPBXi0SM5IK5acwSNhbHaMx9iy9wyDseSNBGbpGXi08EvkGEjz8EgkbrvfYybB28aQuOFGAhszDx4tbBI5xr+BWuo3t/GYSf5tY6gnqAVoixnIlgQDNh4zaaAtCQaEtEj2PCuzBPrFcMYxtmJr2TYJw5lnHjZL49NicDx5842PPXXy/G3MG2++bbOR5zuefPAzPi1gwNgDZ4KihrGBkAYg+EGEmlEwCkbBKBi5AADcNkNwHBajhAAAAABJRU5ErkJggg==","orcid":"","institution":"Huashan Hospital, Fudan University","correspondingAuthor":true,"prefix":"","firstName":"Daojie","middleName":"","lastName":"Xu","suffix":""}],"badges":[],"createdAt":"2025-03-23 12:23:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6288169/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6288169/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11064-025-04462-3","type":"published","date":"2025-06-25T15:57:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":80319831,"identity":"e95cd964-1fcf-47d1-9916-d44818995dee","added_by":"auto","created_at":"2025-04-10 13:15:12","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":221219,"visible":true,"origin":"","legend":"\u003cp\u003ePropofol treatment induces cell apoptosis in the hippocampal dentate gyrus in mice.\u003c/p\u003e\n\u003cp\u003e(A) Survival rate of mice induced by intraperitoneal injection of propofol for 3 days. (B) Quantification of apoptosis in the DG region of P21 mice after continuous intraperitoneal injection of propofol for 3 days. (C) Representative c-c3-positive cell immunofluorescence induced by propofol treatment. Blue, DAPI-stained nuclei. Scale bar, 50 μm. The data are presented as the means ± standard errors. Ctrl: control group; P: propofol group. *\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05; **\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01. n=5 for each group.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6288169/v1/6b93e8fe8640461e4f812335.png"},{"id":80319832,"identity":"7a5edc54-74a6-4d75-a3ea-f1fca853489b","added_by":"auto","created_at":"2025-04-10 13:15:12","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":145539,"visible":true,"origin":"","legend":"\u003cp\u003ePropofol administration led to cognitive dysfunction in adulthood. (A) Performance of the mice in the open field test. Open field test results for the distance traveled (B), mean speed (C), distance traveled in the center (D) and time spent in the center (E). (F) Swimming speed in the Morris water maze training task for the Ctrl and P groups. (G) Latency time in the Morris water maze training task for the Ctrl and P groups. (H) The number of platform crossings during the probe test for the Ctrl and P groups. (I) The time spent in the target zone by the Ctrl and P groups. Ctrl, control group; P, propofol group. The data are presented as the means ± standard errors. *\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05; **\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01. n=12 for each group.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6288169/v1/d55753097d75975c8e0d2bff.png"},{"id":80319110,"identity":"e9e63fce-3d7f-4e22-ba3c-3600373457c0","added_by":"auto","created_at":"2025-04-10 13:07:12","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":45735,"visible":true,"origin":"","legend":"\u003cp\u003ePropofol exposure induced the activation and upregulation of Rac1. (A) The expression of Rac1 and Rac1-GTP (activated Rac1 form) in the dentate gyrus was measured via Western blotting. (B) Quantitative analysis of Rac1-GTP protein expression in the Ctrl and P groups. (C) Quantitative analysis of Rac1 protein expression in the Ctrl and P groups. The data are presented as the means ± standard errors. **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01. n=5 for each group. Ctrl: control group; P: propofol group.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6288169/v1/295428a1f0a127ec200d2124.png"},{"id":80320368,"identity":"bd78f80a-07d7-4884-be5c-cdfb3221df7a","added_by":"auto","created_at":"2025-04-10 13:23:12","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":258719,"visible":true,"origin":"","legend":"\u003cp\u003eInhibiting Rac1 activation attenuated the neuroapoptosis induced by propofol exposure. (A) Representative western blots of Rac1 and Rac1-GTP in each group after administration of the Rac1 agonist CN04-A. (B) Quantification of Rac1-GTP/Rac1 in the dentate gyrus according to western blots. (C) Representative western blots of Rac1 and Rac1-GTP after the application of the Rac1 inhibitor NSC23766 in each group. (D) Quantification of Rac1-GTP/Rac1 in the dentate gyrus according to western blots. (E) Cleaved caspase-3 (c-c3, red) was used to obtain immunofluorescence images of typical coronal dentate gyrus (DG) slices, which were costained with DAPI (blue) to identify apoptotic cells in each group after treatment with the Rac1 agonist or inhibitor. The image magnification is 20×; scale bar, 50 μm. (F) Quantitative comparison of c-c3 in each group after the application of the Rac1 agonist CN04-A. (G) Quantitative comparison of c-c3 in each group after the application of the Rac1 inhibitor NSC23766. The data are presented as the means±standard errors (n=5). *: Compared with the vehicle-treated Ctrl group, *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, ** \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01. #: Compared with the Ctrl group treated with agonist/inhibitor, ##\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01. △: Compared with the P group treated with vehicle, △△ \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01. Ctrl: control group; P: propofol group.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6288169/v1/468d00c090a7ce6128b45c5c.png"},{"id":80319834,"identity":"a15d6b16-a8c8-4ac9-930d-f5ba00763938","added_by":"auto","created_at":"2025-04-10 13:15:12","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":273377,"visible":true,"origin":"","legend":"\u003cp\u003eKnockdown of Rac-1 in the dentate gyrus attenuated neuroapoptosis induced by propofol. (A, C) Representative western blots of Rac1 in the hippocampus after AAV injection. (B. D) Quantification of Rac1 expression in the dentate gyrus according to western blots. (E) Quantitative comparison of c-c3 in each group after Rac1 silencing. Cleaved caspase-3 (c-c3, red) is used to mark the immunofluorescence images of typical coronal dentate gyrus (DG) slices, which are costained with DAPI (blue) to show apoptotic cells, and GFP (green) shows DG cells transfected with adeno-associated virus. The image magnification is 20×; scale bar, 50 μm. (F) Quantification of c-c3 after the silencing of Rac1 in each group. (G) Quantification of c-c3 in each group after the overexpression of Rac1. The data are presented as the means±standard errors (n=5). *: Compared with the Ctrl group treated with the empty virus vector, *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, ** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; #: Compared with the Ctrl group with overexpression/silencing, ##\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01. △: Compared with the P group treated with the empty virus vector, △△\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01. Ctrl: control group; P: propofol group. Mean±SEM (n=5).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6288169/v1/ca2ffdb92114b86462eb48b7.png"},{"id":80320372,"identity":"d200f234-9c31-49db-9de3-805136174b30","added_by":"auto","created_at":"2025-04-10 13:23:13","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":185995,"visible":true,"origin":"","legend":"\u003cp\u003eKnockdown of Rac1 in the dentate gyrus attenuated the cognitive deficits induced by propofol exposure. (A) Trajectory diagram of the open field test. (B) Quantitative comparison of the distance traveled in each group. (C) Quantitative comparison of the mean speed in each group. D Quantitative comparison of distance traveled in the central area in each group. (F) Quantitative comparison of the time spent in the central area in each group. (G) Quantitative comparison of the swimming speed in each group in the Morris water maze test. (H) Quantitative comparison of the escape latency in each group. (I) Qualification of the number of times crossing the target zone in each group. (J) Quantitative comparison of the time in the target zone in each group. m*: Compared with the Ctrl group, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01. Ctrl: control group; P: propofol treatment group; C-shRac1: Rac1-silenced empty virus vector group; ShRac1: Rac1-silenced group. The data are presented as the means ± standard errors. n=12 for each group.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6288169/v1/84b8d3e9ae10be766283267a.png"},{"id":80319121,"identity":"d93587ae-ca5e-4e23-8041-ae170147c89e","added_by":"auto","created_at":"2025-04-10 13:07:13","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":114235,"visible":true,"origin":"","legend":"\u003cp\u003ePropofol affects the expression of Akt pathway proteins through the expression of Rac1. (A) Changes in p-Akt expression after propofol treatment. (B) Quantitative comparison of p-Akt protein expression in each group. (C) Changes in p-Akt expression after the overexpression of Rac1. (D, E) Quantitative comparison of p-Akt protein expression in each group after Rac1 overexpression. (F) Changes in p-Akt expression after Rac1 silencing. (G, H) Quantitative comparison of p-Akt protein expression in each group after Rac1 silencing. Mean ± SEM (n=5). *: Compared with the empty virus vector group, **: \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01. C-ovRac1: Rac1-overexpressing empty virus vector group; OvRac1: Rac1-overexpressing group; C-shRac1: Rac1-silenced empty virus vector group; ShRac1: Rac1-silenced group.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-6288169/v1/0196cabb7110079c9a1c5988.png"},{"id":85686276,"identity":"ee7bcf52-3650-4e51-94ad-5c6f0c4d19cf","added_by":"auto","created_at":"2025-06-30 16:05:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2022635,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6288169/v1/37a85d40-3ce3-47bd-8b65-8d34e1ae27df.pdf"},{"id":80320370,"identity":"4c092778-30d4-4e98-be9b-47d4cb668f43","added_by":"auto","created_at":"2025-04-10 13:23:13","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":1238501,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-6288169/v1/21e15c3e05106ba50350dddb.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Inhibition of Rac1 attenuated propofol-induced neurotoxicity in the hippocampal dentate gyrus in developing mice","fulltext":[{"header":"Background","content":"\u003cp\u003eMillions of children require general anesthesia worldwide because of the need for surgery or medical procedures every year[1]. However, numerous animal studies have demonstrated that early exposure to general anesthetics in the developing brain may cause neuronal apoptosis and neurocognitive impairment[2-5]. Some retrospective human studies also reported that repeated exposure to general anesthesia in children is associated with the development of cognitive and behavioral deficits later in life[6-8]. Consequently, the United States Food and Drug Administration (FDA) has issued a warning emphasizing the potential neurotoxic effects of general anesthetics on the developing brains of children under the age of three and on fetuses when administered to pregnant women (www.fda.gov/Drugs/DrugSafety/ucm532356.htm).\u003c/p\u003e\n\u003cp\u003ePropofol is one of the most commonly used intravenous anesthetic agents in children because of its rapid onset and rapid recovery following discontinuation of administration[9]. Like other general anesthetics, early exposure to propofol also causes neuronal apoptosis in the developing brain, raising serious concerns about the safety of propofol anesthesia in children[10, 11]. Nevertheless, the molecular and cellular mechanisms underlying the effects of early propofol exposure\u0026nbsp;on brain development remain poorly understood. Therefore, investigating the signaling pathways involved in propofol-induced neuronal apoptosis is imperative.\u003c/p\u003e\n\u003cp\u003eRas-related C3 botulinum toxin substrate 1 (Rac1), a small Rho guanosine triphosphate phosphohydrolases (GTPases), is recognized for its responsiveness to environmental stress in cells or organisms. It detects external signals from receptors on the membrane and triggers downstream signaling pathways to perform its physiological functions[12]. Rac1 is vital for regulating several cellular processes, including cytoskeletal structure, oxidative product generation, and gene expression[13]. Specifically, Rac1 plays a crucial role in brain functions such as neuronal migration, synaptic plasticity, and memory formation by regulating actin dynamics in neurons[14]. Irregular Rac1 expression and activity have been noted in various neurological disorders[15]. Recently, the role of Rac1 in neuroapoptosis has attracted increasing attention. Research has indicated that the inhibition of Rac1 activity ameliorates neuronal apoptosis and cognitive deficits caused by cerebral ischemia‒reperfusion in the hippocampal CA1 region of rats[16]. Additionally, neurotrophic factors alleviate propofol-induced neuronal apoptosis in the hippocampus, a process contingent upon Rac1 expression[17]. However, whether Rac1 is involved in propofol-induced neurotoxicity remains unknown.\u003c/p\u003e\n\u003cp\u003eTherefore, the aim of the present work was to determine whether Rac1 participates in propofol-induced neurotoxicity. In our study, repeated exposure to propofol induced widespread apoptosis in the developing dentate gyrus (DG), accompanied by increased expression of Rac1. Modulating Rac1, either through inhibition or overexpression, can mitigate or exacerbate propofol-induced neuronal apoptosis, respectively. Moreover, we found that Rac1 silencing mitigated propofol-induced long-term cognitive dysfunction. These results indicate that Rac1 might be a promising therapeutic target for the clinical treatment of propofol-induced neurotoxicity.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eExperimental animals\u003c/h2\u003e \u003cp\u003eC57BL/6 mice were used. The procedures adhered to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Ethical Committee for Animal Research of Fudan University (No. 201802130S). All the mice were reared in temperature- and humidity-controlled rooms with a 12-h light/dark cycle. Efforts were made to reduce the number of animals used to minimize the number required for statistical accuracy and minimize suffering. Both male and female mice were used in this study.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAnesthesia\u003c/h3\u003e\n\u003cp\u003eThe mice were randomly divided into a control group and a propofol group. In the propofol group, the mice were injected intraperitoneally with 150 mg/kg propofol from P19 to P21 once a day for three consecutive days. After the injection, they were placed on a constant temperature blanket at 37\u0026deg;C for 6 h to prevent hypothermia. On the third day after the last injection, the animals were euthanized 6 h after complete recovery, and samples were collected for western blot and immunofluorescence analysis.\u003c/p\u003e\n\u003ch3\u003eInjection of agonists/inhibitors\u003c/h3\u003e\n\u003cp\u003eThe specific agonist CN04-A (848 \u0026micro;mol/L, 1 \u0026micro;l/side, Cytoskeleton, USA) and the inhibitor NSC23766 (125 \u0026micro;mol/L, 1 \u0026micro;l/side, Tocris, UK) for Rac1 activity were dissolved in ultrapure water and freshly prepared on the day of the experiment. P19 mice were anesthetized via intraperitoneal injection of pentobarbital and secured in a stereotaxic frame. After shaving and disinfecting, the scalp was incised, and the subcutaneous tissue was separated to fully expose the skull. The anterior fontanelle, posterior fontanelle, coronal suture, and sagittal suture were adequately revealed. A cranial drill was subsequently used at the DG site (with the anterior fontanelle as the reference point, ML\u0026thinsp;=\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5 mm, AP = -2 mm, and DV = -2.5 mm) to gently create an opening in the skull for needle insertion. Glass electrode filaments were pulled, and their tips were observed under a microscope. A diameter of 50 \u0026micro;m was left at the tip, prefilled with mineral oil to eliminate air, and connected to a microsyringe (1 ml) via hot melt adhesive. The microsyringe was then slowly injected with 1 \u0026micro;l/side of the solvent into the dentate gyrus of both sides of the mouse, taking over 5 min for the injection. Afterward, the microsyringe was left in place for 5 min, followed by slow withdrawal to prevent reflux. Postoperatively, the area was disinfected, sutured, and marked for subsequent experimental injections.\u003c/p\u003e\n\u003ch3\u003eInjection of Adeno-associated virus\u003c/h3\u003e\n\u003cp\u003eShort hairpin RNA (shRNA) targeting Rac1 was constructed by Obio Technology in Shanghai, China. This shRNA was inserted into an adeno-associated virus (AAV) vector carrying green fluorescent protein (GFP). This shRNA is designed to specifically target mouse neurons, and it can specifically downregulate or upregulate the expression of Rac1. The same vector backbone was used to construct a negative control. For virus brain stereotaxic microinjections in P3 mice, the mice were anesthetized through intraperitoneal injection of pentobarbital and secured in a stereotaxic frame. Then, with a microsyringe (1 ml) connected to a glass electrode with a tip diameter of 50 \u0026micro;m, 1 \u0026micro;l of the virus was slowly injected into the DG on both sides of the mice (with the anterior fontanelle as the reference point, ML\u0026thinsp;=\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0 mm, AP = -1.5 mm, DV = -2 mm) over a period exceeding 5 min. Finally, the microsyringe was left in place for 5 min, followed by slow withdrawal to prevent reflux.\u003c/p\u003e\n\u003ch3\u003eImmunohistochemistry, image acquisition and analysis\u003c/h3\u003e\n\u003cp\u003eSix hours after saline or anesthesia injection, all pups were deeply anesthetized with 0.7% sodium pentobarbital and perfused with 0.9% saline, followed by 4% paraformaldehyde (PFA) in PBS. The brains were dissected and postfixed in 4% PFA/PBS for 4\u0026ndash;6 h at 4\u0026deg;C and equilibrated in 30% sucrose. Coronal sections (30 \u0026micro;m) containing the DG were cut using a Leica CM1950 cryostat (Wetzlar, Germany), and every 5th section was immunostained. The sections were incubated in a blocking solution containing 5% bovine serum albumin (BSA) and 0.5% Triton X-100 for 2 h at 37\u0026deg;C. A primary rabbit monoclonal antibody against cleaved caspase-3 (c-c3, 9661 L, 1:400; Cell Signaling Technology, Beverly, MA, USA) was applied overnight in 0.3% BSA at 4\u0026deg;C. After being washed three times in PBS, the sections were incubated with an Alexa Fluor-conjugated (488 or 568 nm) secondary antibody at 1:500 (Thermo Fisher Scientific, Waltham, MA, USA) for 2 h at 37\u0026deg;C. Nuclei were labeled with 4\u0026prime;,6-diamidino-2-phenylindole (DAPI) (Thermo Fisher Scientific) for 30 min at 37\u0026deg;C.\u003c/p\u003e \u003cp\u003eThe stained slices were scanned via the TissueFAXS Plus system. The number of cleaved caspase-3 (c-c3)-positive cells was quantified. Following the completion of all the image scans, the image data were processed and analyzed via image analysis software (ImageJ, version 1.48 V, USA). Costaining of c-c3 and DAPI nuclear staining was used to identify apoptotic neurons. All the data were subjected to blind analysis via Image-Pro Plus software (Media-Cybernetics, Rockville, MD, USA) under experimental conditions. The cells were considered c-c3 positive if they were larger than the threshold size and displayed detectable cell contours. Approximately 40 c-c3-positive cells were imaged and analyzed per mouse. To analyze the proportion of c-c3-positive cells, all images were thresholded before counting, and colocalization was defined as overlap between thresholded regions. For all the experiments, brain tissue sections from the control and anesthetized mice were coprocessed, including those from the immunostaining, imaging, and analysis steps. Each experiment involved the analysis of 10 image frames from each mouse.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eArterial blood gas measurements\u003c/h2\u003e \u003cp\u003eArterial blood gas was measured in mic at 3 h or 6 h following propofol anesthesia. Arterial blood was obtained via transcardiac aspiration of the left ventricle via a heparinized 32-gauge hypodermic needle. The pH, partial pressure of carbon dioxide (PaCO\u003csub\u003e2\u003c/sub\u003e), PaO\u003csub\u003e2\u003c/sub\u003e, SaO\u003csub\u003e2\u003c/sub\u003e, and HCO\u003csup\u003e3\u0026minus;\u003c/sup\u003e concentrations were measured immediately after arterial blood collection via a portable clinical analyzer (ABL800FLEX, Radiometer, Copenhagen, Denmark).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePull-down assay\u003c/h3\u003e\n\u003cp\u003eTo prepare the tissue lysate, the tissue was homogenized in prechilled 1\u0026times; lysis buffer (#20\u0026ndash;168, Millipore) and placed on ice for 30 min. Afterward, it was centrifuged at 4000 \u0026times; g for 15 min at 4\u0026deg;C, and the supernatant was collected. The sample concentration was determined via the BCA protein assay method. Pak1 is an effector protein that can bind to active Rac1. To detect this interaction, 1 mg of total protein was incubated with 10 \u0026micro;l of PAK-1 PBD agarose beads for 1 h. The agarose beads were subsequently washed three times, resuspended in 40 \u0026micro;l of 2\u0026times; reducing sample buffer and boiled for 5 min. The bound proteins were separated via 12.5% SDS‒PAGE and detected by immunoblotting with a Rac1 antibody (#05‒389, Millipore) to determine the level of GTP-bound Rac1.\u003c/p\u003e\n\u003ch3\u003eWestern blotting\u003c/h3\u003e\n\u003cp\u003eThe mice were deeply anesthetized via i.p. injection of sodium pentobarbital, followed by decapitation, after which the regions encompassing the bilateral DGs were dissected at designated time points and preserved in liquid nitrogen. Next, total proteins were extracted from the DGs by homogenizing the tissues in ice-cold RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China) supplemented with 0.1 mM phenylmethylsulfonyl fluoride-protease inhibitors and phosphatase inhibitors, lysing them on ice for 30 min, and centrifuging them at 4\u0026deg;C for 10 min. Samples were heated at 95\u0026deg;C for 10 min in loading buffer before equal amounts of protein were loaded onto a 10% sodium dodecyl sulfate\u0026ndash;polyacrylamide gel for electrophoretic separation (SDS‒PAGE) and then transferred onto polyvinylidene difluoride membranes at 4\u0026deg;C. After being blocked with 5% nonfat milk for 2 h at room temperature, the membranes were incubated overnight at 4\u0026deg;C with a primary antibody (1:1000 dilution) or β-actin (1:500 dilution, Boster #BM3873, Wuhan, China). The membranes were washed with PBS buffer and incubated with horseradish peroxidase-conjugated secondary anti-rabbit IgG (1:2000 dilution; Abcam #ab6721, Cambridge, MA) for 2 h at room temperature, and the bands were visualized via enhanced chemiluminescence (ECL, Thermo Fisher China, Shanghai, China). The relative levels of the target protein normalized to the level of β-actin were determined via densitometry analysis via ImageJ software (National Institutes of Health, Bethesda, MD, USA).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eMorris water maze\u003c/h2\u003e \u003cp\u003eBehavioral assessments were conducted in a quiet, light-regulated environment. Prior to initiating the water maze experiments, the mice underwent a three-day habituation period to familiarize them with the testing room and experimenters. Both groups of mice were introduced into a circular plastic pool with a diameter of 120 cm, filled with water that had been dyed black. The water temperature was consistently maintained at 23\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C. Visual cues were strategically placed around the perimeter of the pool to assist the mice in locating the submerged platform.\u003c/p\u003e \u003cp\u003eFor each trial, the mice were gently introduced into the pool from one of four preassigned entry points and given 60 s to find a platform submerged 2 cm below the water surface. The platform location was randomized among the four quadrants but remained consistent for each individual mouse during the acquisition phase. If a mouse failed to locate the platform within the allotted time, it was guided to the platform. Once on the platform, the mice remained there for 30 s. At the conclusion of each trial, the animals were removed from the pool, dried with a towel, and returned to their home cages.\u003c/p\u003e \u003cp\u003eThe acquisition phase spanned five consecutive days, with one session per day consisting of four trials, each separated by a 30 min interval. The trials were recorded on video, and the swimming trajectories were analyzed via water maze software. The parameters included the time taken to locate the platform and the swimming speed calculated for each trial and averaged over daily sessions.\u003c/p\u003e \u003cp\u003eA probe test was conducted 24 h after the training phase concluded. During this test, the platform was removed, and the animals were allowed to swim freely for 60 seconds. The time spent in the target zone and the number of taget crossings were calculated to evaluate the long-term memory of the platform's previous location.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eOpen Field Test\u003c/h2\u003e \u003cp\u003eThe open field test is a widely used method for assessing both anxiety-related behaviors and locomotor activity. The test was conducted via a Tru Scan (USA) open field apparatus consisting of a 1-meter diameter square arena equipped with infrared sensors to monitor animal movements in both horizontal and vertical dimensions. The mice were individually placed in the open field for a 30 min observation period. The data collected included the total distance traveled and the mean speed, the distance occurring within the central zone, and the time spent in the central area. The behavioral metrics for each mouse were processed and analyzed via TRU SCAN2.1 software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were performed via GraphPad Prism 6 (GraphPad Software, La Jolla, CA, United States). One-way analysis of variance (ANOVA, comparing three or more conditions) followed by Dunnett\u0026rsquo;s test (all conditions compared to the control) and two-way ANOVA (for comparing two independent variables) were used to assess statistical significance, followed by Bonferroni\u0026rsquo;s or Tukey\u0026rsquo;s multiple comparisons test, as specified in the figure legends. The data are shown as the means\u0026thinsp;\u0026plusmn;\u0026thinsp;SEMs. \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant, and all the conditions significantly different from the controls were reported.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003ePropofol induces cell apoptosis in the dentate gyrus of the hippocampus in developing mice\u003c/h2\u003e \u003cp\u003eTo investigate the impact of propofol exposure on neuronal apoptosis in the DG, C57BL/6 mice were treated with propofol from postnatal days 19 to 21 over a span of three consecutive days. Initially, we assessed the survival rates of mice subjected to various dosages of propofol. Our findings indicated that mice could tolerate doses of 100 mg/kg and 150 mg/kg propofol. However, a marked decrease in the survival rate was observed at a dosage of 200 mg/kg propofol (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Consequently, a dosage of 150 mg/kg was selected for the subsequent experiments. Furthermore, compared with that of the control group, 150 mg/kg propofol exposure significantly increased the number of apoptotic cells in the DG, as measured by quantitative analysis of c-c3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, C).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo evaluate whether propofol anesthesia affects the physiological state of mice, arterial blood gas analysis was performed at 3 h and 6 h after propofol anesthesia. Compared with the control group, propofol anesthesia had no effect on the pH, partial pressure of carbon dioxide (PaCO\u003csub\u003e2\u003c/sub\u003e), partial pressure of oxygen (PaO\u003csub\u003e2\u003c/sub\u003e), bicarbonate (HCO\u003csub\u003e3\u003c/sub\u003e), or oxygen saturation (SaO\u003csub\u003e2\u003c/sub\u003e), suggesting that a dosage of 150 mg/kg propofol did not alter the physiological state of the mice (supplementary Table\u0026nbsp;1).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003ePropofol exposure leads to cognitive dysfunction in adulthood\u003c/h2\u003e \u003cp\u003eIn our previous study, exposure of P21 mice to sevoflurane, a widely used general anesthetic, did not affect spatial or memory ability. However, whether repeated exposure to propofol at P21 affects cognitive impairment remains unknown. We initially explored the impact of propofol exposure on long-term cognitive function in developing mice. C57BL/6 mice were treated with propofol at P19-P21, and learning and memory performance in adulthood (P49\u0026ndash;P55) was subsequently examined via the open field test and Morris water maze test. Compared with the control group, the open field test revealed no statistically significant differences in total distance traveled or average movement speed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA‒C). However, compared with the control treatment, propofol treatment decreased the time spent and the frequency of visits to the central area (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, D, E). Similarly, in the Morris water maze test, propofol treatment significantly decreased the number of times crossing the target zone and shortened the time spent in the target zone (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH, I). There was no significant difference in swimming speed and escape latency between the propofol group and the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF, G). These results showed that propofol exposure causes long-term cognitive dysfunction in developing mice.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eRac1 is activated and upregulated in the dentate gyrus of the hippocampus upon propofol exposure\u003c/h2\u003e \u003cp\u003eRac1 acts as a molecular switch, and upon binding to GTP, it forms an activated Rac1-GTP complex and affects downstream Rac1 signaling pathways[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The activation of Rac1 is crucial for brain development. To determine whether propofol exposure enhances Rac1 activation and expression, we analyzed the expression of Rac1-GTP and Rac1 in the hippocampal dentate gyrus by western blotting. Our results revealed that propofol treatment dramatically increased the expression of Rac1-GTP and Rac1 in the hippocampal dentate gyrus (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-C), indicating that Rac1 is activated and upregulated upon propofol exposure.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eInhibition of Rac1 activation attenuated propofol-induced neuroapoptosis in the DG\u003c/h2\u003e \u003cp\u003eTo identify whether the activation of Rac1 in the DG contributes to propofol-induced neuroapoptosis, the Rac1 agonist CN04-A or the inhibitor NSC23766 was injected into the bilateral DG of P19 mice before propofol exposure. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-B, compared with that in the control group, the expression of Rac1-GTP in the mice treated with CN04-A alone was significantly greater. In mice treated with propofol, CN04-A also significantly increased Rac1-GTP expression. In contrast, the Rac1 inhibitor NSC23766 significantly reduced Rac1-GTP expression in both the control and propofol-treated groups, indicating excellent activation or inhibition of Rac1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC-D). Thereafter, c-c3 immunofluorescence was performed to examine the effects of Rac1 activation or inhibition on propofol-induced neuroapoptosis in the DG. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE-G, compared with propofol alone, CN04-A significantly increased propofol-induced apoptosis. Conversely, treatment with NSC23766 markedly reduced propofol-induced apoptosis. Collectively, these results suggest that Rac1 activation plays a role in the neuroapoptosis triggered by propofol exposure.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eKnockdown of Rac1 mitigated propofol-induced neuroapoptosis in the DG\u003c/h2\u003e \u003cp\u003eTo further examine the role of Rac1 expression in propofol-induced neuroapoptosis in the DG, we injected adeno-associated virus (AAV) carrying small-molecule shRNA-Rac1 for overexpression (ovRac1) and silencing (shRac1), along with a Rac1 overexpression empty AAV vector (C-ovRac1) and a silencing empty AAV vector (C-shRac1), into the DG region of P3 mice. When the mice reached P21, we conducted western blot analysis to assess Rac1 protein expression levels in the hippocampal DG region. The mice were randomly divided into two groups: the control group (Ctrl) and the propofol-treated group (P). Compared with the respective empty vector controls for each group, Rac1 overexpression led to a significant increase in Rac1 expression in both the Ctrl and P groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. A-B). Compared with the respective empty vector controls for each group, Rac1 silencing resulted in a significant decrease in Rac1 expression in both the Ctrl and P groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. C-D), with statistically significant differences. These findings indicate the successful establishment of Rac1 overexpression and silencing interference models. Moreover, our results revealed that Rac1 overexpression increased the number of c-c3-positive neurons in the DG region. Compared with those in the propofol plus empty viral vector group, the number of c-c3-positive neurons was greater in the propofol plus Rac1 overexpression group. Similarly, knocking down Rac1 expression relieved propofol-induced c-c3-positive neurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE-G). Taken together, these findings indicate that Rac1 is involved in propofol-induced cell apoptosis in the hippocampal dentate gyrus.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eRac1 silencing mitigated propofol-induced long-term cognitive dysfunction\u003c/h2\u003e \u003cp\u003eMoreover, we examined the potential involvement of Rac1 in long-term cognitive dysfunction. The mice were randomly divided into a control group (Ctrl group), a propofol group (P group), a C-shRac1 group and a shRac1 group. AAV carrying the small molecule shRNA-Rac1 for silencing (shRac1) or the silencing empty AAV vector (C-shRac1) was administered into the DG region of P3 mice in the shRac1 group and the C-shRac1 group, respectively. The mice in the P, shRac1 and C-shRac1 groups were exposed to propofol from P19 to P21. Behavioral assessments were conducted when the mice in all four groups reached adulthood (postnatal days 49\u0026ndash;55). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, compared with the C-shRac1 group, propofol treatment did not affect the total distance traveled or average movement speed in the open field experiments after the expression of Rac1 was knocked down (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-C). However, the distance traveled and the time spent in the central area were ameliorated after the expression of Rac1 was knocked down (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD, E). In the Morris water maze test, there were no statistically significant differences in swimming speed or escape latency between the shRac1 and C-shRac1 groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF, G). However, compared with those in the P group and the C-shRac1 group, the number of times crossing the target zone in the shRac1 group was significantly greater (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH), and the time in the target zone was significantly greater (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eI). Taken together, these results suggest that Rac1 silencing could mitigate propofol-induced long-term cognitive dysfunction.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003ePropofol treatment decreased Akt expression in DG region cells via Rac1\u003c/h2\u003e \u003cp\u003ePrevious studies have shown that Akt is crucial in the regulation of cell apoptosis, whereas Rac1 exerts a regulatory influence on apoptosis by modulating Akt activation[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. To determine whether propofol affects the expression of Akt proteins through Rac1 in the DG region, we first assessed the expression of p-Akt/Akt following propofol treatment. Our results showed that propofol exposure significantly decreased p-Akt expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA, B). Therefore, we explored the effects of Rac1 overexpression on the expression of Akt following propofol treatment. Compared with that in the control group, the expression of p-Akt significantly decreased in the Rac1-overexpressing group (OvRac1 group) and the propofol plus Rac1-overexpressing group (propofol\u0026thinsp;+\u0026thinsp;OvRac1 group) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC-E). We further examined the effects of Rac1 knockdown on the expression of Akt. We found that knocking down the expression of Rac1 did not affect the expression of p-Akt. However, knocking down the expression of Rac1 significantly increased the expression of p-Akt after propofol treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF-H; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Taken together, these results indicate that propofol decreased Akt expression in DG region cells via Rac1.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eOver the past several decades, numerous animal studies have demonstrated that early exposure to nearly all general anesthetics can induce widespread neuroapopsis in the neonatal brain and adversely affect long-term brain development[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Although various mechanisms have been proposed to be involved in the development of neuroapoptosis and cognitive dysfunction following anesthesia, the underlying mechanism is still elusive. In the present study, we observed that early exposure to propofol resulted in extensive cellular apoptosis within the hippocampal dentate gyrus of mice and revealed that Rac1 plays a vital role in propofol-induced cell apoptosis and, subsequently, long-term cognitive dysfunction.\u003c/p\u003e \u003cp\u003eSince our earlier research indicated that anesthesia-induced neuroapoptosis in the DG of mice was postponed, reaching its peak at postnatal day 21 instead of P7[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], the present study exposed mice to propofol from postnatal days 19 to 21. The mice were exposed to propofol at their most vulnerable time to maximize the influence of DG granule cell apoptosis on their long-term behavior. In various studies focused on neuroapoptosis and long-term behavioral disorders caused by anesthesia in the developing brain, rodents were anesthetized at P4\u0026ndash;P8[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. This is very different from our study. The discrepancy may arise from the fact that different brain areas have distinct windows of susceptibility to neurotoxicity induced by anesthesia. Notably, the CA1 region of the hippocampus showed maximum vulnerability to anesthesia-induced neuroapoptosis approximately one week after birth. The CA1 region is also associated with learning and memory[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. This could explain why P4\u0026ndash;P8 mice are regularly used for evaluating neurotoxicity under anesthesia.\u003c/p\u003e \u003cp\u003eRac1 belongs to the family of classical Rho guanosine triphosphate phosphohydrolases (GTPases), which are recognized for their involvement in various cellular processes, including cytoskeletal organization, the regulation of gene expression, and cell migration[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. However, few studies have investigated the regulatory function of Rac1 in propofol-induced neuroapoptosis in the developing brain. Interestingly, we found that Rac1 was upregulated and activated in the dentate gyrus in P21 mice. To elucidate the role of Rac1 activation in the neuroapoptosis induced by propofol, Rac1 agonists and inhibitors were administered. Immunofluorescence analysis of c-c3 expression in the DG revealed that Rac1 activation significantly potentiated propofol-induced apoptosis, whereas inhibition of Rac1 activity substantially mitigated this apoptotic effect. These findings suggest that propofol might modulate apoptosis in the DG region through the regulation of Rac1 activity. In addition, small molecule shRNA-Rac1-overexpressing and small molecule-silenced adeno-associated viruses were utilized to determine the direct effect of Rac1 expression on propofol-induced apoptosis in the DG. Under physiological conditions, the overexpression of Rac1 resulted in increased apoptosis, whereas the silencing of Rac1 did not significantly affect apoptosis in the DG region. These findings suggest a correlation between elevated Rac1 expression and cell apoptosis in the DG. Following treatment with propofol, Rac1 overexpression further augmented propofol-induced apoptosis in DG region cells. Notably, silencing Rac1 led to a significant reduction in propofol-induced apoptosis in the DG. Collectively, these findings indicate that propofol modulates cell apoptosis in the DG via a Rac1-dependent mechanism.\u003c/p\u003e \u003cp\u003eAs demonstrated in previous reports, Rac1 exerts a regulatory influence on apoptosis by modulating Akt activation[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Rac1 is located primarily in the cytoplasm of cells and plays a crucial role in the regulation of various cellular processes. When involved in cell apoptosis, Rac1 is activated and translocated to the inner surface of the plasma membrane, where it recruits Akt to the membrane. This recruitment allows Akt to undergo phosphorylation, thereby engaging in downstream signaling pathways related to cell growth, apoptosis, and other cellular events[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. In the present work, we found that Rac1 overexpression decreased p-Akt expression regardless of propofol exposure. In addition, knocking down the expression of Rac1 significantly increased the expression of p-Akt after propofol treatment. These results indicate that propofol might decrease Akt expression in DG region cells through Rac1 activation. However, the precise mechanism underlying the interaction between Rac1 and Akt under propofol exposure remains to be elucidated.\u003c/p\u003e \u003cp\u003eOur study demonstrated that repeated propofol exposure from P19 to P21 caused long-term cognitive dysfunction in developing mice, as measured by the open field test and Morris water maze test. These findings were slightly different from those of our previous study, which revealed that sevoflurane exposure at P21 had no effect on spatial learning or memory ability in the Morris water maze test[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. While many studies have indicated that exposing newborn mice to general anesthetics negatively impacts long-term spatial learning and memory, some studies have reported no effect or even an improvement in performance in the Morris water maze test. Newborn rats exposed to 2.6% sevoflurane anesthesia for 2 hours on postnatal days 7, 14, and 21 showed no changes in escape latency or platform crossing frequency in the Morris water maze test during adolescence (P31) or adulthood (P91)[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Neonatal mice (P4\u0026ndash;P6) exposed to 1.8% sevoflurane for 6 hours exhibited superior performance in the training phase of the Morris water maze test[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The inconsistency between studies might result from the various anesthetics administered to the mice, the time they were exposed, or the age of the subjects. Moreover, our study demonstrated that Rac1 not only participates in regulating propofol-induced apoptosis but also in regulating cognitive function impairment. Rac1 silencing mitigated propofol-induced cognitive dysfunction in the open-field test and Morris water maze test. Our findings align with those of previous studies, which have shown that inhibiting Rac1 activity significantly mitigates the hippocampal cell damage and neurocognitive impairments induced by sevoflurane in rats. These results indicate that Rac1 might be a promising therapeutic target for propofol neurotoxicity.\u003c/p\u003e \u003cp\u003eOur study acknowledges certain limitations. Specifically, we have shown that propofol exposure leads to a reduction in Akt expression in cells within the DG region through the regulation of Rac1. However, it remains to be definitively established whether the Rac1‒Akt pathway is directly involved in propofol-induced neuroapoptosis. Additionally, we described the neuroapoptotic effect of propofol exposure on P21 mice, and whether the loss of neurons at P21 extends to cognitive impairment in adulthood needs further confirmation.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn conclusion, our study demonstrated that repeated propofol exposure during the development period of mice induced widespread neuroapoptosis in the DG, accompanied by the activation and upregulation of Rac1. Inhibiting the activation or overexpression of Rac1 attenuated propofol-induced neuroapoptosis. Furthermore, Rac1 silencing mitigated propofol-induced long-term cognitive dysfunction. The current work provides evidence that Rac1 could be a promising therapeutic target for the clinical treatment of neuroapoptosis and cognitive dysfunction induced by propofol.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (82271292, 81730031 to Yingwei Wang), the Foundation of Shanghai Municipal Science and Technology Medical Innovation Research Project (23Y21900600 to Yingwei Wang), the Foundation of Shanghai Municipal Key Clinical Specialty (shslczdzk06901 to Yingwei Wang), the National Natural Science Foundation of China (82371285 to Daojie Xu), the Foundation of Shanghai Municipal Science and Technology Commission (22ZR1409600 to Daojie Xu), and the National Natural Science Foundation of China (82201421 to Qi Wang, 82101272 to Kai Wei).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDaojie Xu, Zhiru Wang, Yingwei Wang and Kai Wei contributed to conception and design of the study; Qi Wang, Yuan Li, Haifeng Duan, Yi Lin, Zhoujing Yang contributed to acquisition and analysis of data; Yuan Li, Daojie Xu, Yingwei Wang and Kai Wei contributed to draft the work or substantively revise it. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analysed during this study are included in this published article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was approved in accordance with the Ethical Committee for Animal Research of Fudan University\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors thank the entire staff of the Department of Anesthesiology, Huashan Hospital, Fudan University for critical discussions of this manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eWalkden GJ, Gill H, Davies NM, Peters AE, Wright I, Pickering AE. Early Childhood General Anesthesia and Neurodevelopmental Outcomes in the Avon Longitudinal Study of Parents and Children Birth Cohort. 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ASN Neurology. 2015;7(2).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"neurochemical-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"nere","sideBox":"Learn more about [Neurochemical Research](https://www.springer.com/journal/11064)","snPcode":"11064","submissionUrl":"https://submission.nature.com/new-submission/11064/3","title":"Neurochemical Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Propofol, Dentate gyrus, Neuronal apoptosis, Cognitive dysfunction, Rac1","lastPublishedDoi":"10.21203/rs.3.rs-6288169/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6288169/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAccumulating evidence from animals has shown that multiple exposures to general anesthetics during brain development may cause extensive neuronal apoptosis and long-term memory impairments. However, the underlying mechanism is still poorly understood. In the present study, C57BL/6 mice were administered propofol at postnatal days 19\u0026ndash;21. We found that propofol exposure significantly increased neuronal apoptosis in the mouse hippocampal dentate gyrus, as evidenced by cleaved caspase-3 (c-c3) immunofluorescence. Additionally, propofol exposure increased the activation and expression of Ras-related C3 botulinum toxin substrate 1 (Rac1) in the dentate gyrus, as shown by western blotting. Modulating Rac1, either through inhibition or overexpression, was found to mitigate or exacerbate propofol-induced neuronal apoptosis, respectively. We further demonstrated that propofol decreased the expression of the antiapoptotic protein phosphorylated serine/threonine kinase Akt via Rac1. The results of the open field and morris water maze tests further revealed that silencing Rac1 alleviated propofol-induced cognitive dysfunction. Our findings demonstrated that propofol exposure induced neuronal apoptosis and long-term cognitive dysfunction in the mouse hippocampal dentate gyrus by regulating Rac1.\u003c/p\u003e","manuscriptTitle":"Inhibition of Rac1 attenuated propofol-induced neurotoxicity in the hippocampal dentate gyrus in developing mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-10 13:07:08","doi":"10.21203/rs.3.rs-6288169/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-04-28T06:06:58+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-27T09:39:54+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"320965650378660752696848123637411738501","date":"2025-04-13T09:13:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"11925830574901819197686217446981643372","date":"2025-04-11T07:06:47+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-08T07:26:01+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"314720078236499875475841295302699799481","date":"2025-03-27T08:20:31+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-03-26T11:16:08+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-24T17:20:57+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-03-24T14:35:32+00:00","index":"","fulltext":""},{"type":"submitted","content":"Neurochemical Research","date":"2025-03-23T12:13:55+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"neurochemical-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"nere","sideBox":"Learn more about [Neurochemical Research](https://www.springer.com/journal/11064)","snPcode":"11064","submissionUrl":"https://submission.nature.com/new-submission/11064/3","title":"Neurochemical Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"de253365-f108-4eff-b007-223dd73a7964","owner":[],"postedDate":"April 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-06-30T16:04:19+00:00","versionOfRecord":{"articleIdentity":"rs-6288169","link":"https://doi.org/10.1007/s11064-025-04462-3","journal":{"identity":"neurochemical-research","isVorOnly":false,"title":"Neurochemical Research"},"publishedOn":"2025-06-25 15:57:00","publishedOnDateReadable":"June 25th, 2025"},"versionCreatedAt":"2025-04-10 13:07:08","video":"","vorDoi":"10.1007/s11064-025-04462-3","vorDoiUrl":"https://doi.org/10.1007/s11064-025-04462-3","workflowStages":[]},"version":"v1","identity":"rs-6288169","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6288169","identity":"rs-6288169","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}