Propofol Attenuates Isoflurane-Induced Neurotoxicity and Cognitive Impairment in Fetal and Offspring Mice : Anesthesia & Analgesia

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Original Research Articles: Original Laboratory Research Report

Propofol Attenuates Isoflurane-Induced Neurotoxicity and Cognitive Impairment in Fetal and Offspring Mice

Nie, Yangyang MD, MS*,†; Li, Shuai MD, PhD*; Yan, Tao MD, PhD*; Ma, Yiming PhD; Ni, Cheng MD, PhD*; Wang, Hongying PhD; Zheng, Hui MD, PhD*

Author Information
doi: 10.1213/ANE.0000000000004955
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Abstract

KEY POINTS

  • Question: Can propofol mitigate the effects of isoflurane anesthesia during pregnancy on neuroinflammation and apoptosis in fetal mice and on cognitive functioning in offspring?
  • Findings: We found that propofol attenuated isoflurane-induced neuroinflammation and apoptosis in fetal mice and cognitive impairment in offspring.
  • Meaning: The finding that propofol mitigates isoflurane-induced neurotoxicity and neurobehavioral deficits should prompt further investigation of anesthesia-induced neurotoxicity in the developing brain.

Inhalation anesthetics can exert neurotoxic effects on the developing brain.1–4 In clinical studies, multiple, but not single, exposures to anesthesia and surgery in children appear to be associated with the development of postoperative neurocognitive disorder.3,5–7 Such effects could result from multiple mechanisms. Indeed, increasing evidence from preclinical studies demonstrates cellular apoptosis, impairment of neurogenesis, and inhibition of synapse formation in brain tissues of young mice exposed to inhaled anesthetics via impaired neurotrophic signaling, mitochondrial dysfunction, neuroinflammation, and interneuron phenotype changes.4 However, the underlying mechanisms and potential treatments remain largely to be determined.

Specifically, isoflurane exposure in pregnant mice can impair spatial memory acquisition and reduce anxiety in offspring.8 Our past studies suggest that sevoflurane administration in pregnant mice causes caspase activation, neuroinflammation, and reduction in the postsynaptic density (PSD)-95 protein levels and synaptophysin levels in the brain tissues of fetal and offspring mice, as well as cognitive impairment in offspring.9 Interestingly, isoflurane can induce mitochondrial-dependent apoptosis.10 Isoflurane can also open the mitochondrial permeability transition pore (mPTP), which decreases mitochondrial membrane potential and leads to neurotoxicity.11–13 However, whether such neurotoxicity can be averted or treated when fetal mice have been exposed remains to be determined.

Propofol, a commonly used intravenous anesthetic for pediatric and obstetric procedures that require anesthesia or sedation, can confer neuroprotective effects.14–16 While isoflurane can induce opening of the mPTP and related apoptosis,13 propofol blocks the mPTP.15 In particular, propofol ameliorates isoflurane-induced neurotoxicity by inhibiting mitochondrial dysfunction in cultured cells.17 However, the in vivo relevance of these in vitro findings has not been performed. Interestingly, both propofol and isoflurane exhibit dose- and time-dependent neurotoxic and neuroprotective effects in a variety of cultured cells and the developing brain in experimental animal models.18,19

The objective of the current studies was to assess whether the administration of propofol could mitigate isoflurane-induced neurotoxicity and cognitive impairment in gestationally exposed fetal and postnatal mice. We hypothesized that propofol could attenuate isoflurane-induced apoptosis, neuroinflammation, synaptic loss, and cognitive impairment. Specifically, we investigated the effects of gestational exposure to propofol on isoflurane-induced neurotoxicity in brain tissues of fetal mice and cognitive impairment of postnatal offspring.

METHODS

Animals

This protocol was approved by the Animal Experiments and Experimental Animal Welfare and Ethics Committee of Medical Research Center of Beijing Chaoyang Hospital. The studies conformed to the National Institutes of Health (Bethesda, MD) guidelines, and we wrote this article according to applicable Animal Research: Reporting In Vivo Experiments (ARRIVE) guidelines. Efforts were made to minimize the number of animals used in the studies. Specific pathogen free (SPF), 3-month-old C57BL/6 mice were provided by the Beijing Vital River Laboratory Animal Technology Co, Ltd. Mice were kept at 22°C–23°C on a 12-hour light/dark cycle in an SPF-grade room. Standard food and water for mice were available ad libitum. Each male mouse was housed in a single cage, and female mice were housed in groups of 4–6 per cage. Mice were habituated to the testing environment for 1 week before the experiments.

Timed Pregnancy

Each female mouse was labeled with a unique earmark and weighted. Pairs of female mice were introduced to 1 male and kept in 1 cage. On the second day, we checked female mice for a vaginal plug as a sign of fertilization before placing the mice back into their original cages. If a vaginal plug was observed, the day was recorded as gestational day 1 (G1). At G15, pregnancy was confirmed based on weight and abdominal growth, and pregnant mice were marked and housed separately.

Mouse Anesthesia

At G15, pregnant mice were randomly assigned to 1 of the following 4 groups (n = 6/group): (1) isoflurane (ISO; Baxter, Glenview, IL) plus control (CTL) 2 group, which received 1.4% isoflurane (a clinically relevant concentration)11 in 100% oxygen for 2 hours plus CTL2; (2) propofol (PROP) plus ISO group, which received 50 mg/kg propofol (Fresenius, Beijing, China) via intraperitoneal (i.p.) injection in 100% oxygen for 30 minutes followed by 1.4% isoflurane in 100% oxygen for 2 hours; (3) PROP plus CTL1 group, which only received 50 mg/kg propofol via i.p. injection in 100% oxygen for 30 minutes followed by 100% oxygen for 2 hours; and (4) CTL1 plus CTL2 group, which received 100% oxygen for 2 hours. We used CTL1 for the control condition of ISO anesthesia, CTL2 for the control condition of PROP. Notably, we purposely did not inject intralipid as the control condition of propofol because this control condition (CTL2) was more like the clinical condition in which people without surgeries do not receive any treatments. Previous studies demonstrated that 50 mg/kg propofol i.p. in both ischemia and inhaled anesthesia mouse models is capable of producing neuroprotective effects.17,20 Further, administration of propofol 30 minutes before isoflurane anesthesia could represent an effective preconditioning window.20 Thus, we treated mice with 50 mg/kg propofol i.p. 30 minutes before isoflurane anesthesia.

All mice received identical airflow in the anesthetizing chamber. The temperature of the chamber was regulated to maintain the rectal temperature at 37°C ± 0.5°C. Mice breathed spontaneously while under anesthesia. Concentrations of oxygen and isoflurane were continuously monitored. Previous studies indicate that 2 hours of exposure to 1.4% isoflurane does not significantly affect blood pressure or blood gas in mice.17,21,22 Following treatment, mice were placed in another chamber with 100% oxygen until the righting reflex was reestablished.

Cognitive Testing of Offspring Mice

Based on previous studies,9 the Morris water maze (MWM) was used to test cognitive functions of gestationally exposed mice (n = 15/group) at postnatal day 31 (P31). Dedicated water maze software (Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China) was used to record and analyze the animal’s trajectory and related parameters. P31 offspring were tested in the MWM 4 times per day for 6 days. A video tracking system recorded mouse swimming motions. Mouse body temperature was maintained by active heating for 2–5 minutes after the MWM test.

Brain Tissue Harvesting and Protein Quantification

Immediately after isoflurane anesthesia administration, a cesarean delivery was performed to extract fetal mice, from which brain tissue was harvested. P31 mice were killed by decapitation, and brain tissue was harvested. Separate groups of mice were used for Western blot analysis and immunohistochemistry studies. For Western blot, harvested brain tissues were homogenized on ice using immunoprecipitation buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM ethylenediaminetetraacetic acid, and 0.5% Nonidet P-40) plus protease inhibitors (1 μg/mL aprotinin, 1 μg/mL leupeptin, and 1 μg/mL pepstatin A). Lysates were collected and centrifuged at 12,000 rpm for 15 minutes, and total protein was quantified with a bicinchoninic acid protein assay kit (Applygen Technologies Inc, Beijing, China).9,13

Western Blot Analysis

Hippocampus tissues of P31 mice were used for Western blotting using previously described methods.12 Due to insufficient volumes of fetal hippocampal tissue, whole cerebral hemispheres of fetal mice were used for Western blot analysis. Interleukin (IL)-6 levels and poly-ADP ribose polymerase (PARP) fragment levels were tested in fetal brain tissues; PSD-95 levels were tested in P31 brains. Mouse monoclonal antibodies against IL-6 (1:1000 dilution; R&D SYSTEMS, Minneapolis, MN), PSD-95 (1:1000; Abcam, Cambridge, MA), PARP (1:1000; Cell Signaling Technology, Danvers, MA), and β-actin (1:10,000; Sigma Aldrich, St Louis, MO) were used. Signal intensity was measured using a Bio-Rad (Hercules, CA) Quantity One imaging program.9,13 Densitometric analysis was conducted using β-actin as a control for loading differences. Protein levels in experimental groups were calculated as percentages of the control group.

Immunohistochemistry

P31 mice were anesthetized with 1.4% isoflurane for 4 minutes and perfused transcardially with heparinized saline followed by 4% paraformaldehyde.11 Pilot studies indicated that delivery of 1.4% isoflurane for 4 minutes provides adequate anesthesia for the perfusion procedure in mice without causing statistically significant changes in blood pressure or blood gases. Brain tissues were removed and stored at 4°C in paraformaldehyde. Fixed P31 mouse brains were dehydrated, cleared, and embedded in paraffin, and 5-µm sections were cut from the brain hemispheres. Deparaffinized and rehydrated sections were treated with heated 0.01 M citrate buffer (pH 6.0) for 10 minutes to unmask antigens. Endogenous peroxidase activity was quenched by 0.3% H2O2 in methanol for 30 minutes, followed by blockade of nonspecific binding with rabbit serum for 30 minutes. The sections were then incubated with a monoclonal antibody against synaptophysin (1:500; Sigma Aldrich, St Louis, MO) in a humidified box at 4°C chamber for 24 hours.23,24 The antibody against synaptophysin was detected using a commercial immunoperoxidase staining kit (Zhongshan Goldbridge BioTech Inc, Beijing, China).25 The sections were incubated with biotinylated secondary goat antirabbit antibody for 30 minutes in a humidified box, followed by the avidin–biotin–peroxidase complex reagent incubation for 30 minutes at room temperature. Bound synaptophysin antibody conjugates were visualized by incubating the sections for 10 minutes in 3,3′-diaminobenzidine (DAB) as a chromogen to develop brown staining and then sections were counterstained with hematoxylin and mounted with glycerol gelatin.23,24 We quantified the immunohistochemistry images using the methods described in previous studies.24,26 Slides were screened with an Olympus BX53 microscope equipped with a camera (Olympus, Tokyo, Japan). Twelve sections from each group (6 mice per group) were subjected to the analysis of the optical density (OD) of immunoreactivity in areas cornu ammonis (CA) 1 and dentate gyrus (DG), but not the counting of the number of individual presynaptic puncta, using ImageJ version 1.38 (National Institutes of Health, Bethesda, MD) by an investigator blinded to the experimental design.11,27 The OD of the corpus callosum on the same section was measured and served as background.24 The synaptophysin expression levels were represented by the size of the average integrated OD values. The average OD values of each group were normalized against the average OD value of the control group to obtain standardized values.

Statistical Analysis

We performed a power analysis based on the previous study.9 Assuming a mean difference of 30 seconds (30 vs 60) for the escape latency in the MWM,9 a standard deviation (SD) of 18 in the control arm, an SD of 10 in the anesthesia arm, and a sample size of 15 per arm will lead to >90% power to detect a difference in the behavioral changes using a 2-sided t test with 5% type 1 error. A sample size of 6 per arm will lead to >90% power to detect a mean difference in the biochemistry changes, assuming a mean difference of 150% (100% vs 250%) in the IL-6 levels, an SD of 15 in the control arm, and an SD of 20 in the anesthesia arm.9 Data on escape latency are expressed as mean ± standard error of the mean (SEM). Data on other variables are expressed as mean ± SD. Platform crossing data were not normally distributed (tested by D’Agostino and Pearson normality test) and are thus expressed as median and interquartile range (IQR). Two-way analysis of variance (ANOVA) with repeated measures was used to analyze differences in learning curves (based on escape latency) between groups in the MWM. Multiple comparisons of escape latency in the MWM were adjusted using a Bonferroni correction. Mann–Whitney test was used to determine the difference between groups on platform crossing times. PSD-95, synaptophysin, IL-6, and PARP levels are presented as a percentage of the control group. A two-way ANOVA was used to assess the interaction of propofol with isoflurane anesthesia and to test the hypothesis that propofol could attenuate isoflurane-induced neuroinflammation and neurotoxicity. If the main effects were statistically significant, post hoc comparisons were conducted by a Bonferroni correction. P value <.05 was considered statistically significant. IBM SPSS Statistics Version 21 (International Business Machines Corp) and Prism 6 software (Graph Pad Software, Inc, La Jolla, CA) were used to analyze the data.9

RESULTS

Propofol Mitigated Isoflurane-Induced Cognitive Impairment in Gestationally Exposed Offspring

Pregnant mice at G15 were treated with CTL1 plus CTL2, CTL1 plus PROP, ISO plus CTL2, or ISO plus PROP. Offspring were delivered at G21, and cognitive functions were tested in the MWM from P31 to P36.

Two-way ANOVA with repeated measures for escape latency (the time that each mouse takes to reach a platform during reference training) in the MWM test showed a statistically significant interaction between day (P31 to P36) and group (F = 2.43, P = .0024). Isoflurane anesthesia increased the escape latency as compared to the offspring from dams in CTL1 + CTL2 (F = 78.96, P < .0001; Figure 1A). Platform crossing times (the number of times that each mouse crossed the location of an absent platform at the end of reference training) of offspring gestationally exposed to isoflurane anesthesia were significantly reduced as compared to the offspring from dams in CTL1 + CTL2 (ISO + CTL2: median = 1, IQR = 1 vs CTL1 + CTL2: median = 7, IQR = 6, U = 10.5; P < .0001; Figure 1B).

F1
Figure 1.:
Results of MWM tests. A, Exposure of pregnant dams to 1.4% isoflurane for 2 h at G15 increases the escape latency time of offspring at P31, measured during swimming in MWM as compared to the control condition. *Statistically significant difference in escape latency between the ISO + CTL2 group and CTL1 + CTL2 group. B, Exposure of pregnant dams to 1.4% isoflurane for 2 h at G15 reduces platform crossing times of offspring mice delivered by the dams swimming in the MWM as compared to the control condition. C and D, Propofol 50 mg/kg via intraperitoneal injection alone did not significantly alter escape latency or platform crossing times of offspring mice as compared to the control condition. E, Propofol 50 mg/kg via intraperitoneal injection before exposure to 1.4% isoflurane for 2 h at G15 mitigated the isoflurane-induced increase in escape latency in offspring. **Statistically significant difference in escape latency between ISO + CTL2 group and ISO + PROP group. F, Propofol pretreatment mitigated the isoflurane-induced reduction in platform crossing times. CTL indicates control; G15, gestational day 15; ISO, isoflurane; MWM, Morris water maze; P31, postnatal day 31; PROP, propofol.

Propofol alone did not significantly alter the escape latency or platform crossing times of P31 offspring as compared to CTL1 + CTL2 (Figure 1C, D). These findings indicate that propofol administration alone to dams did not adversely affect cognitive function in offspring.

Moreover, propofol pretreatment of dams mitigated the isoflurane-induced increase in escape latency (F = 34.91, P < .0001; Figure 1E) and reduction in platform crossing times in offspring (ISO + PROP: median = 4, IQR = 2, vs ISO + CTL2: median = 1, IQR = 1, U = 15.5, P < .0001; Figure 1F).

Taken together, these data suggest that propofol may attenuate cognitive dysfunction effects associated with isoflurane exposure during gestation.

Propofol Mitigated Isoflurane-Induced Reduction in PSD-95 and Synaptophysin Levels in the Hippocampus of P31 Offspring

Immunoblotting showed that isoflurane exposure resulted in less visible bands representing PSD-95 in the hippocampus compared to the control condition. However, propofol pretreatment before isoflurane anesthesia produced more visible PSD-95 bands in the hippocampus as compared to isoflurane anesthesia (Figure 2A). Quantification of protein bands showed that isoflurane anesthesia during pregnancy significantly reduced PSD-95 levels in the hippocampus compared with controls (ISO + CTL2: 30.76% ± 2.03% vs CTL1 + CTL2: 100.8% ± 2.25%, t = 39.85, P < .0001). Propofol pretreatment before isoflurane anesthesia in dams mitigated reduced PSD-95 levels in the hippocampus of P31 offspring mice (ISO + PROP: 49.79% ± 3.43% vs ISO + CTL2: 30.76% ± 2.03%, t = 10.83, P< .0001). Propofol alone did not alter the PSD-95 level compared to controls (CTL1 + PROP: 96.41% ± 4.01% vs CTL1 + CTL2: 100.8% ± 2.25%, t = 2.474, P = .1346). Two-way ANOVA indicated an interaction between propofol and isoflurane, and propofol mitigated the isoflurane-induced reduction in PSD-95 levels (F = 88.55, P < .0001; Figure 2B).

F2
Figure 2.:
Western blot analysis of PSD-95. A, Western blot shows that exposure of pregnant dams to 1.4% isoflurane for 2 h at G15 decreases hippocampal PSD-95 levels in P31 offspring as compared to the control condition. Propofol alone does not significantly alter hippocampal PSD-95 levels. However, pretreatment of dams with propofol before isoflurane exposure mitigates isoflurane-induced decreases in PSD-95 levels of P31 offspring. B, Quantification of Western blots shows that gestational isoflurane exposure decreases PSD-95 levels in the hippocampus at P31 as compared to the control condition. Propofol pretreatment before isoflurane exposure mitigates the isoflurane-induced reduction in PSD-95 levels at P31. *P < .0001 vs CTL1 + CTL2 group. **P < .0001 vs ISO + CTL2 group. CTL indicates control; G15, gestational day 15; ISO, isoflurane; N.S., not significant; PROP, propofol; PSD, postsynaptic density.
F3
Figure 3.:
Immunohistochemical analysis of synaptophysin. A, Representative images of synaptophysin immunostaining in the hippocampus of each group. B, The enlargement of black frame areas of A representing CA1 and DG of each group, respectively. Synaptophysin density is decreased in CA1 region and DG in P31 offspring mice after pregnant dams exposed to 1.4% isoflurane for 2 h. Meanwhile, propofol pretreatment before anesthesia restores synaptophysin reduction induced by isoflurane. C and D, Quantification of synaptophysin staining. *P < .0001 vs CTL1 + CTL2 group. **P < .0001 vs ISO + CTL2 group. CA indicates cornu ammonis; CTL, control; DG, dentate gyrus; ISO, isoflurane; N.S., not significant; PROP, propofol.

Consistently, isoflurane anesthesia administration in dams decreased synaptophysin-positive staining in CA1 region and DG in the hippocampus of offspring as compared to controls. Furthermore, propofol pretreatment before isoflurane anesthesia attenuated isoflurane-induced synaptophysin-positive staining decrease in both CA1 region and DG (Figure 3A, B). Quantification of staining showed that isoflurane anesthesia during pregnancy significantly reduced synaptophysin levels in CA1 region (ISO + CTL2: 57.08% ± 4.90% vs CTL1 + CTL2: 100.6% ± 2.20%, t = 22.56, P < .0001) and DG (ISO + CTL2: 56.47% ± 3.76% vs CTL1 + CTL2: 99.76% ± 1.09%, t = 31.94, P < .0001) in the hippocampus of P31 offspring. Propofol pretreatment significantly mitigated this isoflurane-induced reduction in synaptophysin levels in CA1 region (ISO + PROP: 85.57% ± 2.97% vs ISO + CTL2: 57.08% ± 4.90%, t = 14.78, P < .0001) and DG (ISO + PROP: 85.05% ± 1.87% vs ISO + CTL2: 56.47% ± 3.76%, t = 21.09, P < .0001), meanwhile propofol alone did not alter synaptophysin levels in CA1 region and DG in the hippocampus of P31 offspring. Two-way ANOVA indicated an interaction between propofol and isoflurane, and propofol mitigated the isoflurane-induced reduction in synaptophysin levels in CA1 region (Figure 3C) and DG (Figure 3D). These results suggest that isoflurane anesthesia in pregnant mice induced synaptic loss in the hippocampus of P31 offspring, which might underlie cognitive impairments observed by behavioral testing. However, propofol pretreatment attenuated isoflurane-induced synaptic loss in the hippocampus of offspring.

Propofol Attenuated Isoflurane-Induced Neuroinflammation in Fetal Mice

F4
Figure 4.:
Western blot analysis of IL-6. A, Western blot shows that exposure of pregnant dams to 1.4% isoflurane for 2 h at G15 increases brain IL-6 levels in fetuses as compared to the control condition. Propofol alone does not significantly alter fetal brain IL-6 levels. However, pretreatment with propofol mitigates isoflurane-induced increases in IL-6 levels. B, Quantification of protein bands in A. *P < .0001 vs CTL1 + CTL2 group. **P < .0001 vs ISO + CTL2 group. CTL indicates control; G15, gestational day 15; IL, interleukin; ISO, isoflurane; N.S., not significant; PROP, propofol.

Immunoblotting for IL-6 showed that gestational exposure to isoflurane induced more visible bands for IL-6 in the brain tissues of fetuses compared to controls. Propofol pretreatment in pregnant mice before isoflurane anesthesia induced less visible IL-6 bands in brain tissues in fetal mice compared to isoflurane anesthesia (Figure 4A). Quantification of protein bands indicated that isoflurane exposure significantly increased IL-6 levels in fetal brains compared to controls. Propofol pretreatment attenuated the isoflurane-induced increase in IL-6 levels, while propofol alone did not alter IL-6 levels. Two-way ANOVA indicated an interaction between propofol and isoflurane, and propofol mitigated the isoflurane-induced increase in IL-6 levels (F = 48.18, P < .0001; Figure 4B).

Propofol Attenuated Isoflurane-Induced Caspase-3 Activation in Fetal Mice

F5
Figure 5.:
Western blot analysis of PARP. A, Western blot shows that exposure of pregnant dams to 1.4% isoflurane for 2 h at G15 increases fetal brain PARP levels compared to the control condition. Propofol alone does not significantly alter PARP levels. However, pretreatment with propofol mitigates isoflurane-induced increases in PARP levels in the fetal brain. B, Quantification of the protein bands in A. *P < .0001 vs CTL1 + CTL2 group. **P < .0001 vs ISO + CTL2 group. CTL indicates control; ISO, isoflurane; N.S., not significant; PARP, poly-ADP ribose polymerase; PROP, propofol.

Using quantitative Western blot analysis, we observed that isoflurane exposure induced more visible bands of PARP fragments in fetal brain tissue compared to controls. However, propofol pretreatment before isoflurane exposure induced less visible bands of PARP fragments compared to isoflurane anesthesia alone (Figure 5A). Quantification of bands indicated that isoflurane anesthesia during pregnancy significantly increased PARP fragment levels in fetal brain tissue (Figure 5B). Propofol pretreatment significantly attenuated the increase in PARP levels (Figure 5B), while propofol alone did not alter PARP levels. Two-way ANOVA indicated an interaction between propofol and isoflurane, and propofol mitigated the isoflurane-induced increase in PARP levels (F = 58.10, P < .0001; Figure 5B). These data suggest that propofol pretreatment before inhaled isoflurane anesthesia in pregnant mice can attenuate isoflurane-induced caspase activation and neuroinflammation in exposed fetuses.

DISCUSSION

With the increase in infant and maternal exposure to anesthesia during the development of the central nervous system (CNS),28 the safety of anesthesia exposure for developing brain has become a significant health concern.2,3,29

Isoflurane anesthesia in pregnant mice at G15, which corresponds with the neurogenic period during the second trimester, caused learning and memory impairments in P31 offspring (Figure 1). Further, gestationally exposed to isoflurane induced neurotoxicity in offspring, as evidenced by reduced levels of PSD-95 (Figure 2), a marker for postsynaptic membranes,30 and synaptophysin (Figure 3), a membrane-bound protein of synaptic vesicles located in the presynaptic membrane of CNS,31 in the hippocampus. Dams administered isoflurane at G15, during a key period of synaptic development, causing lower levels of synaptophysin and PSD-95, suggesting synaptic loss, might underlie the observed cognitive impairment at P31. Isoflurane exposure also induced neuroinflammation in fetuses, marked by higher levels of IL-6 (Figure 4) and increased caspase-3 activation represented by high levels of PARP fragments in brains (Figure 5). Previous studies showed that reduction in PSD-95 in the hippocampus of P31 offspring induced by inhalation anesthesia during gestation is dependent on increased IL-6 levels.9 Taken together, our data indicate that isoflurane anesthesia during pregnancy induces neuroinflammation and neurotoxicity and causes synaptic loss, leading to cognitive impairment in offspring mice.

Propofol is an intravenous anesthetic commonly used in clinical settings for anesthesia induction in children >3 years old and maintenance in children >2 months old. We selected propofol as a pretreatment drug for its clinical relevance. Propofol pretreatment indeed demonstrated neuroprotective effects. I.P. administration of 50 mg/kg propofol before isoflurane anesthesia in dams mitigated isoflurane-induced changes in fetal brain IL-6 (Figure 4) and PARP (Figure 5) levels, as well as PSD-95 (Figure 2) and synaptophysin levels in P31 offspring (Figure 3), and alleviated cognitive impairment in P31 offspring (Figure 1). In contrast, exposure to 50 mg/kg propofol alone neither increased IL-6 levels in fetal brains (Figure 4) nor affected cognitive function in P31 offspring (Figure 1). These data suggest that propofol might exert neuroprotective effects through anti-inflammatory activity, rescuing isoflurane-induced synaptic loss, and mitigating cognitive impairment.

Notably, the current study demonstrated that i.p. administration to 50 mg/kg propofol alone at G15 appears not to increase proinflammation levels, damage the integrity of synapses, or impair cognitive function. Previous studies found that propofol has dose-dependent32,33 dual effects of neuroprotection14–16 and neurotoxicity3,32–34 on CNS. We speculate that the dual effects of propofol might also be related to the administration route, subjects, windows of treatments, and observation. On the other hand, isoflurane has also been demonstrated to have dual effects of neuroprotection and neurotoxicity, depending on the dose and duration used. Isoflurane at short exposure inhibited cell or brain damage induced by isoflurane itself for prolonged use.19 Our observed neuroprotective effect of propofol on isoflurane-induced neuroinflammation might be related to the dose, timing, and processing duration of pretreatments. We demonstrated that offspring, gestationally exposed to 50 mg/kg propofol i.p. 30 minutes before inhaled 1.4% isoflurane for 2 hours, had better performance in the MWM test than those exposed to isoflurane alone. Further, results showed that levels of synaptophysin and PSD-95 in the propofol pretreatment group were higher than those in the isoflurane group. In light of MWM results, these findings on hippocampal infrastructure suggest that propofol preconditioning before isoflurane anesthesia might have a protective effect on CNS, alleviating neuroinflammatory and neurotoxic effects induced by isoflurane anesthesia.

These findings could have clinical implications for the exposure of fetuses and infants. However, the underlying mechanisms by which propofol exerts this neuroprotective effect remain unclear. Isoflurane induces neuroinflammation associated with the nuclear factor (NF)-κB signaling pathway activation.35 NF-κB, a transcription factor, is the main regulator of the proinflammatory cytokine IL-6, which has been linked to cognitive impairment.11,36 Isoflurane can enhance transcription of NF-κB35 and, as a result, increase IL-6 levels. In animal models of ischemic brain, propofol induces neuroprotection by regulating Toll-like receptor (TLR) 4/NF-κB signaling.37 Considered together, propofol might inhibit TLR4 and downregulate transcription of NF-κB, thereby reducing proinflammatory cytokine IL-6 overproduction to exert anti-inflammatory and neuroprotective effects. Further, in vivo and in vitro studies are warranted to determine whether isoflurane or other inhalation anesthetics can activate TLR4/NF-κB signaling or additional proinflammation pathways to trigger the production of proinflammatory cytokines, such as IL-6. Additionally, research is needed to determine whether or how propofol regulates proinflammation signaling pathways involving TLR4/NF-κB, reduces proinflammatory cytokines other than IL-6, and mitigates isoflurane-induced neuroinflammation and neurotoxicity. Nevertheless, the current findings should lead to further investigation in animals and humans to assess whether propofol can mitigate neurotoxicity and neurobehavioral deficits induced by inhalation anesthetics, for example, isoflurane and sevoflurane.

The current findings illustrate the effects of isoflurane anesthesia during pregnancy on behavioral changes in offspring and the possible underlying cellular mechanisms. Considering that volatile anesthetics have common mechanisms linking them to impaired cognitive performance,4,38 in future research, we will examine the long-term effects of isoflurane exposure in utero (also including sevoflurane and desflurane) on cognitive function. We demonstrated the neuroprotective effect of 1 dose of propofol (50 mg/kg) as a pretreatment for isoflurane anesthesia. Given the dose-dependent dual effects of propofol, the findings should be interpreted with caution. Future studies are needed to assess gradient dose, processing timing window, and durations of propofol exposure to determine the optimal combination to achieve a neuroprotective effect.

In conclusion, our data demonstrate that isoflurane anesthesia in pregnant mice at a clinically relevant dose resulted in adverse effects on fetuses and offspring. Fetal effects include increased IL-6 levels and activated caspase-related apoptosis in brains, while postnatal effects include reduced PSD-95 and synaptophysin levels and impaired cognitive function at age 31 days. These results suggest that gestationally exposed to isoflurane might induce neuroinflammation and neurotoxicity, leading to cognitive impairments. On the other hand, propofol, a commonly used intravenous anesthetic, might rescue isoflurane-induced cognitive impairment by attenuating isoflurane-induced neuroinflammation and neurotoxicity. These findings suggest the need for further investigation of anesthesia-induced neurotoxicity in developing brain and possible treatment strategies.

ACKNOWLEDGMENTS

The authors are grateful for technical assistance from Professor Anshi Wu, Department of Anesthesiology, Beijing Chaoyang Hospital, Capital Medical University, Beijing, China.

DISCLOSURES

Name: Yangyang Nie, MD, MS.

Contribution: This author helped conduct the experiments, analyze the data, and draft the manuscript.

Name: Shuai Li, MD, PhD.

Contribution: This author helped conduct the experiments.

Name: Tao Yan, MD, PhD.

Contribution: This author helped conduct the experiments.

Name: Yiming Ma, PhD.

Contribution: This author helped conduct the experiments.

Name: Cheng Ni, MD, PhD.

Contribution: This author helped analyze the data and edit the manuscript.

Name: Hongying Wang, PhD.

Contribution: This author helped design the experiment and analyze the data.

Name: Hui Zheng, MD, PhD.

Contribution: This author helped design the experiment, analyze the data, and edit the manuscript.

This manuscript was handled by: Robert Whittington, MD.

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