Isoflurane-induced Neurotoxicity Research Articles

HSP90 Inhibition Attenuated Isoflurane-Induced Neurotoxicity in Mice and Human Neuroglioma Cells.

Isoflurane, a widely used inhalation anesthetic in clinical practice, is associated with an increased risk of neuronal injury. Heat shock protein 90 (HSP90) plays a crucial role in maintaining neuronal homeostasis under stress conditions; however, its role during isoflurane exposure remains poorly understood. In this study, we aimed to investigate the protective effects of HSP90 inhibition and explore the regulatory mechanisms underlying these effects during isoflurane exposure. We found that the HSP90 inhibitor 17-N-allylamino-17-demethoxygeldanamycin (17 AAG) has great protective effects in mitigating isoflurane-induced ferroptosis of mouse hippocampus and cultured neuronal cells. We focused on the activity of the crucial protein GPX4 in ferroptosis and found that 17 AAG exerted protective effects, preserving the physiological GPX4 activity under isoflurane exposure; further, 17 AAG restored the protein level of GPX4. Further, we observed that the chaperone-mediated autophagy (CMA) pathway was activated; 17 AAG also mediated GPX4 degradation under isoflurane exposure. Additionally, it interfered with the formation of complexes between HSP90 and Lamp-2a, inhibiting CMA activity, followed by the blockade of GPX4 degradation, further affecting the isoflurane-induced ferroptosis. Based on these findings, we proposed HSP90 inhibition as a protective mechanism against isoflurane-induced ferroptosis in neurons.

Alfaxalone Alleviates Neurotoxicity and Cognitive Impairment Induced by Isoflurane Anesthesia in Offspring Rats.

The anesthetic isoflurane can cause neurotoxicity in fetuses and offspring of rats, affecting their neurodevelopment. However, the underlying mechanisms and therapeutic targets of isoflurane-induced neurotoxicity remain to be identified. Alfaxalone (ALF) is a steroid anesthetic. Steroids have been reported to have neuroprotective effects. This study aimed to investigate whether ALF could alleviate the isoflurane-induced neurotoxicity in fetuses and offspring of rats. On gestation day 15 (G15), the pregnant SD rats were randomly assigned to 4 groups: control 1 (CTL1) + control 2 (CTL2), isoflurane (ISO) + CTL2, CTL1 + ALF, and ISO + ALF. To analyze the changes in the expression levels of inflammatory cytokines, apoptotic factors, and synaptophysin, the brain tissues from the G15 fetuses and offspring at postnatal day 7 (P7), postnatal day 14 (P14), and postnatal day 31 (P31) were collected. The newborn neurons in the rats' offspring at P7, P14, and P31 were counted using immunofluorescence techniques. The Morris water maze (MWM) test was performed to assess the learning and memory abilities of P31 offspring rats. ALF significantly alleviated the isoflurane-induced increase in the expression levels of inflammatory cytokines and apoptotic factors, such as interleukin (IL)-6 (ISO + CTL2 versus ISO + ALF: 5.133 ± 0.739 versus 1.093 ± 0.213, P < .001) and Caspase-3 (6.457 ± 0.6 versus 1.062 ± 0.1, P < .001) in the G15 fetuses. In P31 offspring rats, the expression levels of synaptophysin (0.719 ± 0.04 versus 1.068 ± 0.072, P < .001) and the number of newborn neurons in the dentate gyrus of the hippocampus were significantly lower in the ISO + CTL2 group as compared to those in the ISO + ALF group (118 ± 6 versus 140 ± 7, P < .001). These changes also occurred in the rat offspring at P7 and P14. In the MWM test, the escape latency of CTL1 + ALF group rats was significantly lower than that of ISO + ALF group rats (41 ± 6 versus 31 ± 7, P < .001) at P31. Based on these findings, this study suggested that isoflurane exposure during pregnancy in rats could cause neuroinflammation and death of embryos as well as impairment of cognitive function in the offspring rats. ALF can be used to counteract the negative effects of isoflurane.

Acutely increased aquaporin-4 exhibits more potent protective effects in the cortex against single and repeated isoflurane-induced neurotoxicity in the developing rat brain

Damage to hippocampus, cerebellum, and cortex associated with cognitive functions due to anesthetic-induced toxicity early in life may cause cognitive decline later. Aquaporin 4 (AQP4), a key protein in waste clearance pathway of brain, is involved in synaptic plasticity and neurocognition. We investigated the effects of single and repeated isoflurane (Iso) anesthesia on AQP4 levels and brain damage. Postnatal-day (P)7 Wistar albino rats were randomly assigned to Iso or Control (C) groups. For single-exposure, pups were exposed to 1.5% Iso in 30% oxygenated-air for 3-h at P7 (Iso1). For repeated-exposure, pups were exposed to Iso for 3 days, 3-h each day, at 1-day intervals (P7 + 9 + 11) starting at P7 (Iso3). C1 and C3 groups received only 30% oxygenated-air. Based on HE-staining and immunoblotting (Bax/Bcl-2, cleaved-caspase3 and PARP1) analyses, Iso exposures caused a higher degree of apoptosis in hippocampus. Anesthesia increased 4-hydroxynonenal (4HNE), oxidative stress marker; the highest ROS accumulation was determined in cerebellum. Increased inflammation (TNF-α, NF-κB) was detected. Multiple Iso-exposures caused more significant damage than single exposure. Moreover, 4HNE and TNF-α contributed synergistically to Iso-induced neurotoxicity. After anesthesia, higher expression of AQP4 was detected in cortex than hippocampus and cerebellum. There was an inverse correlation between increased AQP4 levels and apoptosis/ROS/inflammation. Correlation analysis indicated that AQP4 had a more substantial protective profile against oxidative stress than apoptosis. Remarkably, acutely increased AQP4 against Iso exhibited a more potent neuroprotective effect in cortex, especially frontal cortex. These findings promote further research to understand better the mechanisms underlying anesthesia-induced toxicity in the developing brain.

Maprotiline ameliorates isoflurane-induced microglial activation via regulating triggering receptor expressed in myeloid cells 2 (TREM2)

ABSTRACT Isoflurane-induced neurotoxicity has attracted much interest. Recent studies suggest that isoflurane causes microglial activation, resulting in an inflammatory response and microglial insult. Maprotiline is a novel drug that has been licensed as an antidepressant with considerable anti-inflammatory activity. However, it is still unknown whether maprotiline possesses a protective effect against isoflurane-induced microglial insult. Here, we found that maprotiline ameliorated isoflurane-caused reduction in BV2 microglial cell viability and lactate dehydrogenase (LDH) release. Maprotiline mitigated isoflurane-induced oxidative stress by inhibiting reactive oxygen species (ROS) production and increasing superoxide dismutase (SOD) activity. Isoflurane-induced expression and production of inflammatory markers including tumor necrosis factor (TNF-α), interleukin (IL)-1β, cyclooxygenase‐2 (COX-2), and prostaglandin E2 (PGE2) were decreased in maprotiline-treated cells. Maprotiline inhibited the mRNA and protein levels of Iba1, a marker of microglial activation, in isoflurane-induced BV2 cells. Maprotiline treatment restored isoflurane-induced reduction of TREM2 in BV2 microglial cells. In addition, the knockdown of TREM2 abolished the beneficial effects of maprotiline against isoflurane. Collectively, maprotiline exerted protective effects against isoflurane-caused oxidative stress, inflammatory response, and cell injury via regulating TREM2. These findings show that maprotiline prevented the isoflurane-induced microglial activation, indicating that maprotiline might be used as an optimal therapeutic agent for preventing the isoflurane-caused neurotoxicity.

Tetraethylammonium chloride reduces anaesthetic-induced neurotoxicity in Caenorhabditis elegans and mice

BackgroundIf anaesthetics cause permanent cognitive deficits in some children, the implications are enormous, but the molecular causes of anaesthetic-induced neurotoxicity, and consequently possible therapies, are still debated. Anaesthetic exposure early in development can be neurotoxic in the invertebrate Caenorhabditis elegans causing endoplasmic reticulum (ER) stress and defects in chemotaxis during adulthood. We screened this model organism for compounds that alleviated neurotoxicity, and then tested these candidates for efficacy in mice. MethodsWe screened compounds for alleviation of ER stress induction by isoflurane in C. elegans assayed by induction of a green fluorescent protein (GFP) reporter. Drugs that inhibited ER stress were screened for reduction of the anaesthetic-induced chemotaxis defect. Compounds that alleviated both aspects of neurotoxicity were then blindly tested for the ability to inhibit induction of caspase-3 by isoflurane in P7 mice. ResultsIsoflurane increased ER stress indicated by increased GFP reporter fluorescence (240% increase, P<0.001). Nine compounds reduced induction of ER stress by isoflurane by 90–95% (P<0.001 in all cases). Of these compounds, tetraethylammonium chloride and trehalose also alleviated the isoflurane-induced defect in chemotaxis (trehalose by 44%, P=0.001; tetraethylammonium chloride by 23%, P<0.001). In mouse brain, tetraethylammonium chloride reduced isoflurane-induced caspase staining in the anterior cortical (–54%, P=0.007) and hippocampal regions (–46%, P=0.002). DiscussionTetraethylammonium chloride alleviated isoflurane-induced neurotoxicity in two widely divergent species, raising the likelihood that it may have therapeutic value. In C. elegans, ER stress predicts isoflurane-induced neurotoxicity, but is not its cause.

Fraxetin suppresses reactive oxygen species-dependent autophagy by the PI3K/Akt pathway to inhibit isoflurane-induced neurotoxicity in hippocampal neuronal cells.

Isoflurane, a common volatile anesthetic, has been widely used to provide general anesthesia in operations. However, exposure to isoflurane may cause widespread neurotoxicity in the developing animal brain. Fraxetin, a natural coumarin derivative extracted from the bark of Fraxinus rhynchophylla, possesses versatile pharmacological properties including anti-oxidative, anti-inflammatory, and neuroprotective effects. However, the effect and action mechanism of fraxetin on neurotoxicity induced by isoflurane are unknown. Reactive oxygen species (ROS) generation, cell viability, lactate dehydrogenase (LDH) release, and apoptosis were estimated by 2',7'-dichlorofluorescin-diacetate (DCFH-DA) staining, MTT, LDH release, and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) staining assays, respectively. The protein levels of light chain 3 (LC3)-I, LC3-II, p62, protein kinase B (Akt), and phosphorylated Akt (p-Akt) were detected by western blot analysis. Isoflurane induced ROS, LDH release, apoptosis, and autophagy, but inhibited the viability in HT22 cells, which were overturned by fraxetin or ROS scavenger N-acetyl-L-cysteine. Fraxetin suppressed isoflurane-induced PI3K/Akt inactivation in HT22 cells. PI3K/Akt inactivation by LY294002 resisted the effects of fraxetin on isoflurane-induced autophagy and autophagy-modulated neurotoxicity in HT22 cells. In conclusion, fraxetin suppressed ROS-dependent autophagy by activating the PI3K/Akt pathway to inhibit isoflurane-induced neurotoxicity in hippocampal neuronal cells.

Biochanin A protects SH-SY5Y cells against isoflurane-induced neurotoxicity by suppressing oxidative stress and apoptosis

Biochanin A (BCA) is a natural organic O-methylated isoflavone with a variety of pharmacological effects, and has been reported to have neuroprotective properties. Here, we explored whether BCA protects neurocytes against isoflurane-induced neurotoxicity and investigated the underlying mechanism. Cell viability was tested by cell counting kit-8 and lactate dehydrogenase release assays. Apoptosis was evaluated by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and caspase-3/7 activity assays. Superoxide dismutase (SOD) and catalase (CAT) activities and levels of glutathione (GSH) and malondialdehyde (MDA) were measured to assess oxidative stress. Expression of nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1) and NAD(P)H quinone oxidoreductase (NQO1) was determined by western blotting. Treatment with BCA significantly attenuated the reduction of cell viability induced by isoflurane in SH-SY5Y cells. In addition, BCA treatment reversed isoflurane-induced SOD and CAT activity reduction, GSH level decline and MDA level increase. Isoflurane-induced apoptosis was also attenuated by treatment with BCA. The increase in nuclear Nrf2, HO-1 and NQO1 expression induced by isoflurane was amplified by treatment with BCA. These inhibitory effects of BCA on isoflurane-induced oxidative stress, viability reduction and cell apoptosis were attenuated in Nrf2 knockdown SH-SY5Y cells. Our findings indicate that BCA protects SH-SY5Y cells against isoflurane-induced neurotoxicity via inducing the Nrf2/ARE pathway.

Genistein Attenuates Isoflurane-Induced Neuroinflammation by Inhibiting TLR4-Mediated Microglial-Polarization in vivo and in vitro.

BackgroundIsoflurane, a widely used anesthetic in surgery, has been found to induce neurotoxicity. In parallel, genistein is thought to attenuate isoflurane-induced neurotoxicity, although underlying molecular mechanisms are still unclear. In this study, we studied the protective effects of genistein on isoflurane-induced neuroinflammation in rats and BV2 cells.MethodsSprague-Dawley rat pups were exposed to 0.75% isoflurane for 6 hours at postnatal day 7 (P7), and genistein (20, 40, or 80 mg/kg/day) or saline administered from P3 to P15. Hippocampal single-cell suspensions were prepared and apoptosis analyzed by flow cytometry. mRNA expression was determined by RT-qPCR, while protein expression was assessed using Western blot, immunochemistry and immunofluorescence. TLR4 was knocked-out in BV2 cells through CRISPR-Cas9.ResultsGenistein treatment reduced isoflurane-induced apoptosis and inflammation in rat hippocampus. Importantly, genistein promoted M2 and suppressed M1 microglia polarization in rat hippocampus after stimulation with isoflurane. In addition, genistein reduced isoflurane-induced protein expression levels of TLR4, MyD88, TRAF6, p-TAK1, p-p38, p-ERK, p-IκBα and p-NF-κB in rat hippocampus. In BV2 cells exposed to isoflurane, genistein treatment decreased IL-1β, TNF-α, IL-6 and IL-8 mRNA expressions, promoted M2 and suppressed M1 microglia polarization. Similarly, genistein also decreased TLR4 protein levels in isoflurane-induced BV2 cells. However, genistein did not affect CD16, iNOS, CD206 and Arg1 protein levels in TLR4-KO BV2 cells exposed to isoflurane.ConclusionGenistein attenuates isoflurane-induced neurotoxicity by inhibiting TLR4-mediated microglial inflammation in vivo and in vitro.

Repeated isoflurane exposures of neonatal rats contribute to cognitive dysfunction in juvenile animals: the role of miR-497 in isoflurane-induced neurotoxicity.

Isoflurane anesthesia at the period of brain development can lead to neurotoxicity and long-term cognitive impairment. This study aimed to investigate the role of miR-497 on isoflurane-induced neurotoxicity. Neonatal rats (P7) were subject to isoflurane for 2 h at P7, P9, and P11. MiR-497 and neuron apoptosis were evaluated in hippocampal tissue by qRT-PCR and western blot. Fear conditioning test and Morris water maze were performed to determine cognitive function. The cell viability of isolated hippocampal neuronal cells exposed to isoflurane was measured using MTT test. The regulation of phospholipase D1 (PLD1) by miR-497 in isolated hippocampal neuronal cells was evaluated by luciferase reporter assays and western blot. Immunohistochemistry and TUNEL staining were employed to examine the PLD1 expression and neuronal cell apoptosis in hippocampus of neonatal rats, respectively. Repeated isoflurane anesthesia led to neurons' apoptosis and long-term cognitive impairment. Isoflurane exposure led to apoptosis and viability reduction in hippocampal neuronal cells. MiR-497 was observed to be upregulated after isoflurane exposure both in vivo and in vitro. Knockdown of miR-497 attenuated isoflurane-induced neuronal cells apoptosis and viability reduction. Furthermore, PLD1 was predicted and then validated as a novel target of miR-497. miR-497 could negatively regulate PLD1 by binding to its 3'-untranslated region. Downregulation of PLD1 was also observed after isoflurane exposure in neonatal rat hippocampus and hippocampal primary neuronal cell cultures. Induction of miR-497 was involved in isoflurane anesthesia-induced cognitive impairment and neuronal cell apoptosis by targeting PLD1. miR-497 may be a novel potential mechanism in isoflurane-induced neurotoxicity so that our findings provide new insight into a better and understanding of the clinical application of isoflurane.

Ferroptosis contributes to isoflurane-induced neurotoxicity and learning and memory impairment

Ferroptosis is a novel type of programmed cell death, which is different from apoptosis and autophagic cell death. Recently, ferroptosis has been indicated to contribute to the in vitro neurotoxicity induced by isoflurane, which is one of the most common anesthetics in clinic. However, the in vivo position of ferroptosis in isoflurane-induced neurotoxicity as well as learning and memory impairment remains unclear. In this study, we mainly explored the relationship between ferroptosis and isoflurane-induced learning and memory, as well as the therapeutic methods in mouse model. Our results indicated that isoflurane induced the ferroptosis in a dose-dependent and time-dependent manner in hippocampus, the organ related with learning and memory ability. In addition, the activity of cytochrome c oxidase/Complex IV in mitochondrial electron transport chain (ETC) was increased by isoflurane, which might further contributed to cysteine deprivation-induced ferroptosis caused by isoflurane exposure. More importantly, isoflurane-induced ferroptosis could be rescued by both ferroptosis inhibitor (ferrostatin-1) and mitochondria activator (dimethyl fumarate), which also showed effective therapeutic action against isoflurane-induced learning and memory impairment. Taken together, our data indicate the close association among ferroptosis, mitochondria and isoflurane, and provide a novel insight into the therapy mode against isoflurane-induced learning and memory impairment.

MiR-191 downregulation protects against isoflurane-induced neurotoxicity through targeting BDNF

Background Isoflurane inhalation can cause nerve damage, and miR-191 is abnormally expressed in nerve crush injuries. This study aimed to explore the effect of miR-191 on isoflurane-induced cognition impairment and neurotoxicity in vivo and in vitro, as well as its potential mechanism. Methods Sprague-Dawley male rats and primary hippocampal neurons were applied and exposed to 2% isoflurane. The level of miR-191 was regulated both in vitro and in vivo to investigate the role of miR-191 in isoflurane-induced neurotoxicity. Morris water maze assay was used to evaluate the neurological function of rats. The level of miR-191 was measured by qRT-PCR. CCK-8 assay and Flow cytometry were applied to detect the cell viability and apoptosis. Dual luciferase reporter gene detection was used for the target gene analysis. Results miR-191 was up-regulated in the hippocampal tissues of rats exposed to isoflurane. Downregulation of miR-191 ameliorated isoflurane-induced cognition impairment, including the increase of the neurological score and the escape latency, and the decrease of the time spent in the original quadrant for the rats exposed to isoflurane. Isoflurane exposure inhibited hippocampal neuron viability and promoted cell apoptosis, which was reversed by down-regulation of miR-191. BDNF is a target gene of miR-191. Conclusions Isoflurane causes some neurotoxic effect which is mediated through miR-191 abnormal expression targeting BDNF. Downregulation of miR-191 has a protective role against isoflurane-induced neurotoxicity, regulates the vitality and slows down neuronal cell apoptosis.

Long Non-Coding RNA BACE1-AS Modulates Isoflurane-Induced Neurotoxicity to Alzheimer's Disease Through Sponging miR-214-3p.

Isoflurane, an anesthetic, can aggravate the progression of Alzheimer's disease (AD). Long non-coding RNA β-secretase 1 (BACE1)-antisense transcript (BACE1-AS) and miR-214-3p arerelated to AD progression. Nevertheless, it is unclear whether BACE1-AS is involved in the development of isoflurane-mediated AD via miR-214-3p. Amyloid beta peptide (Aβ) was employed to construct the AD cell model. The expression of BACE1-AS and miR-214-3p in the plasma of AD patients and SK-N-SH and SK-N-AS cells treated with Aβ and isoflurane was assessed through quantitative reverse transcription polymerase chain reaction (qRT-PCR). The proliferation and apoptosis of Aβ-treated SK-N-SH and SK-N-AS cells were determined via 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) or flow cytometry assays, respectively. Protein levels of B cell lymphoma 2 (Bcl-2), Bcl-2-associated X (Bax), CyclinD1, microtubule-associated protein A1/1B-light chain3 (LC3 I/LC3 II), p62 and Beclin1 were detected via western blot analysis. The relationship between BACE1-AS and miR-214-3p was verified by dual-luciferase reporter assay. We found that BACE1-AS was upregulated and miR-214-3p was downregulated in the plasma of AD patients and SK-N-SH and SK-N-AS cells treated with Aβ and isoflurane. Both BACE1-AS depletion and miR-214-3p augmentation restored the suppression of proliferation and the facilitation of apoptosis and autophagy of Aβ-treated SK-N-SH and SK-N-AS cells induced by isoflurane. Importantly, BACE1-AS acted as a sponge for miR-214-3p. Additionally, miR-214-3p silencing reversed the influence of BACE1-AS knockdown on isoflurane-mediated proliferation, apoptosis and autophagy in Aβ-induced SK-N-SH and SK-N-AS cells. In conclusion, BACE1-AS aggravated isoflurane-induced neurotoxicity to AD via sponging miR-214-3p.

The protective effect of trilobatin against isoflurane-induced neurotoxicity in mouse hippocampal neuronal HT22 cells involves the Nrf2/ARE pathway

Long-term exposure to isoflurane may induce long-term developmental neurotoxicity and cognitive impairments in the neonatal brains. Trilobatin, a leaf extract from the Chinese traditional sweet tea Lithocarpus polystachyus Rehd, possesses various biological properties including anti-inflammatory and anti-oxidant properties. Our study aimed to explore the neuroprotective effect of trilobatin on isoflurane-induced neurotoxicity in mouse hippocampal neuronal HT22 cells. The effects of trilobatin on cell viability, LDH release, apoptosis, and caspase-3/7 activity in isoflurane-induced HT22 cells were explored by CCK-8, LDH release assay, flow cytometry analysis, and caspase-3/7 activity assay, respectively. Oxidative stress was evaluated by measuring the levels of reactive oxygen species (ROS) and malonyldialdehyde (MDA) and activities of superoxide dismutase (SOD) and catalase (CAT). The expression of nuclear erythroid-2 related factor 2 (Nrf2), nuclear Nrf2, heme oxygenase-1 (HO-1), and NAD(P)H: quinone oxidoreductase 1 (NQO1) was determined by western blot and qRT-PCR. Results suggested that exposure to isoflurane significantly reduced cell viability and increased LDH release, apoptotic rate and caspase-3/7 activity in HT22 cells, which were abolished by trilobatin. Trilobatin reversed isoflurane-induced increase of ROS and MDA levels and reduction of SOD and CAT activities in HT22 cells. Additionally, trilobatin promoted the nuclear translocation of Nrf2 as well as the mRNA and protein expression of HO-1 and NQO1 in HT22 cells exposed to isoflurane. Nrf2 knockdown attenuated the effects of trilobatin on isoflurane-induced viability reduction, LDH release, apoptosis, and oxidative stress in HT22 cells. Overall, trilobatin protected HT22 cells against isoflurane-induced neurotoxicity via activating the Nrf2/antioxidant response element (ARE) pathway.

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

Anesthesia in pregnant rodents causes neurotoxicity in fetal and offspring rodents. However, the underlying mechanisms and targeted treatments remain largely to be determined. Isoflurane and propofol are among commonly used anesthetics. Thus, we set out to investigate whether propofol can mitigate the isoflurane-induced neurotoxicity in mice. Pregnant C57BL/6 mice at gestational day 15 (G15) were randomly assigned to 4 groups: control, isoflurane, propofol, and isoflurane plus propofol. Levels of interleukin (IL)-6 and poly-ADP ribose polymerase (PARP) fragment were measured in the brains of G15 embryos, and levels of postsynaptic density (PSD)-95 and synaptophysin were determined in the hippocampal tissues of postnatal day 31 (P31) offspring using Western blotting and immunohistochemical staining. Learning and memory functions in P31 offspring were determined using a Morris water maze test. Isoflurane anesthesia in pregnant mice at G15 significantly increased brain IL-6 (222.6% ± 36.45% vs 100.5% ± 3.43%, P < .0001) and PARP fragment (384.2% ± 50.87% vs 99.59% ± 3.25%, P < .0001) levels in fetal mice and reduced brain PSD-95 (30.76% ± 2.03% vs 100.8% ± 2.25%, P < .0001) and synaptophysin levels in cornu ammonis (CA) 1 region (57.08% ± 4.90% vs 100.6% ± 2.20%, P < .0001) and dentate gyrus (DG; 56.47% ± 3.76% vs 99.76% ± 1.09%, P < .0001) in P31 offspring. Isoflurane anesthesia also impaired cognitive function in offspring at P31. Propofol significantly mitigated isoflurane-induced increases in brain IL-6 (117.5% ± 10.37% vs 222.6% ± 36.45%, P < .0001) and PARP fragment (205.1% ± 35.99% vs 384.2% ± 50.87%, P < .0001) levels in fetal mice, as well as reductions in PSD-95 (49.79% ± 3.43% vs 30.76% ± 2.03%, P < .0001) and synaptophysin levels in CA1 region (85.57% ± 2.97% vs 57.08% ± 4.90%, P < .0001) and DG (85.05% ± 1.87% vs 56.47% ± 3.76%, P < .0001) in hippocampus of P31 offspring. Finally, propofol attenuated isoflurane-induced cognitive impairment in offspring. These findings suggest that gestational isoflurane exposure in mice induces neuroinflammation and apoptosis in embryos and causes cognitive impairment in offspring. Propofol can attenuate these isoflurane-induced detrimental effects.

Vitexin improves neuron apoptosis and memory impairment induced by isoflurane via regulation of miR-409 expression

Anesthetics, such as isoflurane, sevoflurane, ketamine, and desflurane, are commonly used in clinics. Specifically, isoflurane is one of the most commonly used inhalational anesthetics, which can be used in surgery patients of all ages, including children. The aim of the study was to investigate the mechanisms of vitexin against isoflurane-induced neurotoxicity. Reference memory testing was performed for 5 days (4 trials, 2 per day) before anesthesia. Reversal testing was performed on the 3rd day after anesthesia. The cell viability and apoptosis of PC-12 cells were detected using MTT and TUNEL assays, respectively. Enzyme-linked immunosorbent assay (ELISA) kits were used to measure serum tumor necrosis factor α (TNF‑α), interleukin 6 (IL‑6), glutathione (GSH), and superoxide dismutase (SOD) concentrations. The concentration of reactive oxygen species (ROS) was detected using ROS measurement. Expression of miR-409 was determined using quantitative reverse-transcription polymerase chain reaction (qPT-PCR). Protein expression levels were detected using western blotting. Rats treated with isoflurane showed significant increases in the escape latency periods (ELP) and the apoptosis of hippocampus neuron cells; this effect was reversed by 3 mg/kg or 10 mg/kg of vitexin (p < 0.05). Further testing showed that isoflurane could significantly decrease the cell viability and increase the apoptosis of PC-12, the expression of inflammatory cytokines (TNF‑α and IL‑6) and ROS (p < 0.05). However, these results were reversed by 10/100 μM of vitexin. In addition, vitexin could significantly increase the expression of miR-409 (p < 0.05). Further studies showed that overexpression of miR-409 could significantly promote the effect of vitexin on isoflurane-induced neurotoxicity (p < 0.05). Finally, overexpression miR-409 could significantly increase the expression of p-AMPK/t-AMPK and p-GSK3β/t-GSK3β. Vitexin has protective effects against isoflurane-induced neurotoxicity by targeting miR-409 and the AMPK/GSK3β pathway.

Isoflurane-induced expression of miR-140-5p aggravates neurotoxicity in diabetic rats by targeting SNX12.

MicroRNAs (miRNAs) are widely known as critical regulators in isoflurane-induced neurotoxicity during the development of brain. Moreover, isoflurane could aggravate cognitive impairment in diabetic rats. The present study was designed to investigate the role and mechanism of miR-140-5p on isoflurane-induced neurotoxicity in diabetic rats. Firstly, a diabetic rat model was established by injection of streptozotocin (STZ) and identified by Morris water maze test. The result indicated that isoflurane treatment exacerbated STZ-induced cognitive impairment, as demonstrated by increase of the latency to the platform and decrease of the proportion of time spent in the target quadrant. Secondly, miR-140-5p was up-regulated in diabetic rats treated with isoflurane. Functional assays revealed that knockdown of miR-140-5p attenuated neurotoxicity in diabetic rats, which was shown by a decrease of the latency to the platform and an increase of the proportion of time spent in the target quadrant. Mechanistically, we demonstrated that miR-140-5p directly bonded to SNX12 (sorting nexin 12). At last, the neuroprotective effect of miR-140-5p knockdown against isoflurane-aggravated neurotoxicity in diabetic rats was dependent on up-regulation of SNX12 and inhibition of cell apoptosis. In summary, these meaningful results demonstrated the mitigation of miR-140-5p knockdown against isoflurane-aggravated neurotoxicity in diabetic rats via SNX12, suggesting a novel target for neuroprotection in diabetes under isoflurane treatment.

Neuroprotection of miR-214 against isoflurane-induced neurotoxicity involves the PTEN/PI3K/Akt pathway in human neuroblastoma cell line SH-SY5Y

Isoflurane, one of the commonly used inhalation anesthetics worldwide in clinical practice, may generate substantial risks of neurotoxicity in the developing brains. The present study aimed to illustrate the effects and underlying mechanisms of miR-214 on isoflurane-induced neurotoxicity in human neuroblastoma cell line SH-SY5Y. SH-SY5Y cells were transfected with miR-214 or miR-con alone or in combination with pcDNA empty vector or pcDNA-PTEN in the presence of 3% isoflurane and incubated for 48 h. Cell viability, lactate dehydrogenase (LDH) release, apoptosis, and caspase-3/7 activity were evaluated using CCK-8, LDH release assay, flow cytometry analysis, and caspase-3/7 activity assay, respectively. The superoxide dismutase (SOD), glutathione (GSH), and malondialdehyde (MDA) activities were measured using commercial kits. miR-214 expression and alterations of the phosphatase and tensin homolog (PTEN)/phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) pathway were detected by qRT-PCR and Western blot, respectively. The interaction between miR-214 and PTEN was explored by luciferase reporter assay. We found that isoflurane exposure induced neurotoxicity in SH-SY5Y cells, as evidenced by the reduced cell viability, increased LDH release, apoptotic rate, caspase-3/7 activity, and oxidative stress levels. Moreover, isoflurane exposure decreased the expression of miR-214 and affected the PTEN/PI3K/Akt pathway in SH-SY5Y cells. miR-214 overexpression significantly suppressed isoflurane-induced viability reduction, LDH release, apoptosis and oxidative stress, as well as inactivation of the PI3K/Akt pathway in SH-SY5Y cells. Interestingly, PTEN was identified as a target of miR-214. Moreover, PTEN upregulation blocked the effects of miR-214 on isoflurane-induced neurotoxicity in SH-SY5Y cells. In conclusion, miR-214 protected against isoflurane-induced neurotoxicity in SH-SY5Y cells via regulation of PI3K/Akt pathway by targeting PTEN, contributing to better understanding the underlying mechanisms of anesthetics-induce neurotoxicity.

Dexmedetomidine pretreatment attenuates isoflurane-induced neurotoxicity via inhibiting the TLR2/NF-κB signaling pathway in neonatal rats

Isoflurane is a commonly used inhalational anesthetic that can induce neurotoxicity, while Dexmedetomidine (Dex) has significant neuroprotective effects. In our study, we explored the effects of Dex on isoflurane-induced neurotoxicity through the TLR2/NF-κB signaling pathway. Seven-day old neonatal Sprague-Dawley rats pretreated with 25, 50, 75 μg/kg Dex were exposed to 0.75% isoflurane for 6 h. Spatial learning and memory abilities were detected by Morris water maze test. Ultrastructure of hippocampal neurons, neuronal apoptosis, and the levels of TLR2/NF-κB signaling pathway-related factors were determined. Besides, TLR2 agonist Pam3CSK4 or NF-κB inhibitor BAY11-7082 was injected to further validate the involvement of TLR2/NF-κB signaling following Dex treatment. Consequently, we found isoflurane inhalation resulted in increased cell apoptosis, inflammation and TLR2/NF-κB signaling pathway activation, and decreased PSD95 expression and spatial learning and memory abilities. Dex led to decreased inflammation, improved neuronal structure and viability in rats as well as enhanced spatial learning and memory abilities of rats, and it inactivated the TLR2/NF-κB signaling pathway. Additionally, Pam3CSK4 injection reversed the protective effects of Dex on isoflurane-induced neurotoxicity. In conclusion, this study provided evidence that Dex could alleviate isoflurane-induced neurotoxicity through inhibition of the TLR2/NF-κB signaling pathway. The findings may offer novel insights for the clinical usage of anesthetics.

NR2B receptor- and calpain-mediated KCC2 cleavage resulted in cognitive deficiency exposure to isoflurane

BackgroundDuring brain development, volatile anesthetic can rapidly interfere with physiologic patterns of dendritic development and synaptogenesis and impair the formation of precise neuronal circuits. KCC2 plays vital roles in spine development and synaptogenesis through its Cl- transport function and structural interactions with the spine cytoskeleton protein 4.1 N. The aim of this study was to dissect the mechanism of volatile anesthetics, which impair dendritic development and synaptogenesis via mediation of KCC2 cleavage. MethodsWesternblotting was employed to assess the expression change of NR2B, NR2A, calpain-1, calpain-2, KCC2, and 4.1 N protein of rat (PND 5). Co-immunoprecipitation was applied to demonstrate the interaction between KCC2 and 4.1 N protein. Long-term cognitive deficiency was assessed by MWM. Lentivirus-calpain-2 was administered by hippocampus stereotaxic injection. ResultsThere was a significant increase in the level of NR2B instead of NR2A exposure to isoflurane. Calpain-2 was excessively activated via NR2B after 6 h of isoflurane exposure. The expression of plasmalemmal KCC2 and 4.1 N protein was significantly decreased treated with isoflurane. The isoflurane group showed longer traveled distance, prolonged escape latency, less time spent in the target quadrant, and decreased platform crossings. Pretreatment with ifenprodil and downregulated calpain-2 expression significantly alleviated these neurotoxicity responses and cognitive deficiency after isoflurane exposure. ConclusionsA significant increase in NR2B, excessive activation of calpain-2 and increased cleavage of plasmalemmal KCC2, are involved in isoflurane-induced neurotoxicity and long-term cognitive deficiency. Blocking NR2B and calpain-2 activity significantly attenuated these responses. The KCC2 cleavage mediated by NR2B and calpain-2 is a major determinant of isoflurane-induced long-term cognitive deficiency.

MicroRNA-24 alleviates isoflurane-induced neurotoxicity in rat hippocampus via attenuation of oxidative stress.

Several miRNAs have been recently suggested as potential therapeutic targets for anesthesia-related diseases. This study was carried out to explore the biological roles of miR-24 in isoflurane-treated rat hippocampal neurons. Isoflurane was used to induce neurotoxicity in a rat model. Gain- and loss-of-function of miR-24 was performed, and the size and Ca2+ permeability of mitochondria, as well as cell proliferation and apoptosis, and levels of oxidative-stress-related factors were measured both in vivo and in vitro. Dual luciferase reporter gene assays were used to identify the target relationship between miR-24 and p27kip1. In this study, isoflurane treatment decreased miR-24 expression, after which, levels of neuron apoptosis and oxidative-stress-related factors were elevated and neuron viability was reduced. Over-expression of miR-24 inhibited oxidative damage and neuronal apoptosis in hippocampal tissues, and suppressed the size and Ca2+ permeability of mitochondria of hippocampal neurons. miR-24 enhanced the viability of rat hippocampal neurons by targeting p27kip1. To conclude, this study demonstrated that miR-24 attenuates isoflurane-induced neurotoxicity in rat hippocampus via its antioxidative properties and inhibiting p27kip1 expression.