These effects recommend that the neurocognitive impairment subsequent fuel exposure was not likely owing to hypoxia/hypoventilation or pathoglycemia.120685-11-2 citationsIn the open up-field take a look at, there was no major variation between O2-enriched air group and isoflurane group in the full distance and the percentage of time spent in the middle of the open up-industry(t = .143, df = 22, p = .888 t = -.027, df = 22, p = .979), which recommended repeated neonatal isoflurane exposure experienced no important influence on locomotor activity and panic degree (Fig 1A and 1B). In the current examine, we assessed the neurocognitive perform of mice working with CFC trials. During CFC training, all mice exhibited few freezing behavior ahead of the footshock was presented there was no major big difference amongst O2-enriched air team and isoflurane group (t = .197, df = 22, p = .845 Fig 1C). During CFC testing, the isoflurane group showed a important reduction in freezing time (t = three.334, df = 22, p = .003), which recommended that their skill to forming fear-connected memory was impaired by isoflurane (Fig 1C). We assessed the degrees of histone acetylation in the CA1 hippocampal area at various time points immediately after CFC training. The benefits confirmed that acetylation of H3K9, H3K14, H4K5, and H4K12 at 1 h following CFC education were substantially increased relative to baseline stage at naive affliction in the mice exposed to thirty% O2-enriched air (H3K9 F(3,20) = 10.358, p<0.001 H3K14 F(3,20) = 10.728, p<0.001 H4K5 F(3,20) = 14.288, p<0.001 H4K12 F(3,20) = 12.494, p<0.001 Multiple comparisons H3K91 h VS control, p = 0.023 H3K141 h VS control, p = 0.036 H4K51 h VS control, p = 0.009 H4K121 h VS control, p = 0.023), but there were no differences among baseline, 15 min, and 24 h after CFC training (all p>.05 in multiple comparisons, Fig 2B). In the isoflurane-uncovered mice, H3K9, H3K14, and H4K5 acetylation had been likewise improved 1 h soon after CFC teaching (H3K9 F(3,20) = 12.322, p<0.001 H3K14 F(3,20) = 6.841, p = 0.002 H4K5 F(3,20) = 13.006, p<0.001 Multiple comparisons H3K91 h VS control, p = 0.011 H3K141 h VS control,effect of repeated neonatal isoflurane exposure on behavioral testing. (A) Mice that received repeated neonatal exposure to 0.75% isoflurane displayed normal traveled total distance in Open-field tests. (B) Mice that received repeated neonatal exposure to 0.75% isoflurane displayed normal affective state (anxiety level), which was measured by the percentage of time spent in the center of open-field. (C) Mice that received repeated neonatal exposure to 0.75% isoflurane displayed decreased freezing time ( p<0.01 versus control) during contexual fear conditioning (CFC) test. The control group inhaled 30% O2-enriched air during the neonatal period.Repeated neonatal exposures to isoflurane induce H4K12 acetylation dysregulation in the CA1 hippocampal region in response to CFC training. (A) Representative images of western blots showing histone acetylation levels in the CA1 hippocampal region at 15 min,1 h, and 24 h after CFC training. Control mice were not subjected to CFC. (B) Quantification of the immunoblots from mice that received repeated exposure to 30% O2-enriched air. p<0.05 and p<0.01 versus control. (C) Quantification of the immunoblots in mice that received repeated neonatal exposure to isoflurane. p<0.05 and p<0.01 versus control. Mean (SD) values are shown p = 0.014 H4K51 h VS control, p = 0.002), while H4K12 acetylation was unaffected (F(3,20) = 0.070, p = 0.975 Fig 2C).To examine whether the HDACi TSA was capable of attenuating isoflurane-induced neurocognitive impairment, we divided the mice into four groups: an air+vehicle group subjected to three neonatal exposures to O2-enriched air and injected with DMSO (vehicle) 2 h before CFC training, an air+TSA group with neonatal exposure to O2-enriched air injected with TSA, an ISO+vehicle group that received repeated neonatal exposures to 0.75% isoflurane and vehicle injection 2 h before CFC training, and an ISO+TSA group that received isoflurane exposures and TSA injection. During the CFC training phase, there were no differences in freezing times among the four groups (F(3,44) = 0.606, p = 0.615 Fig 3A). When re-exposed to the CFC chamber 24 hours later, the ISO+vehicle group exhibited a significant reduction in freezing behavior compared with the other three groups (F(3,44) = 6.545, p = 0.001 Fig 3A). TSA only facilitated memory formation in mice with neonatal isoflurane exposure(Multiple comparisons: ISO+vehicle VS ISO+TSA, p = 0.037 air+vehicle VS air+TSA, p = 0.992) (Fig 3A). At 1 h after CFC training, western blotting analysis showed that TSA increased H3K9, H3K14, H4K5, and H4K12 acetylation in the hippocampal CA1 area in all groups (all p<0.05 in multiple comparisons). H4K12 acetylation in the ISO+vehicle group was significantly decreased compared with the air+vehicle group (F(3,20) = 26.392, p<0.001 ISO+vehicle VS air+vehicle in multiple comparisons, p = 0.012 Fig 3C)c-Fos belongs to the activator protein-1 family of transcription factors and is an immediate early gene (IEG). Many previous studies have demonstrated that c-Fos expression is rapidly elevated by various experiential stimuli including conditioned and unconditioned aversive stimuli [16], sensory stimuli (e.g., auditory, visual, tactile, olfactory)[170], and various learning task [219]. Assessing c-Fos provides information about synaptic plasticity and neuronal activation required for long-term memory formation [27, 30]. In our study, compared with the air +vehicle group, the ISO+vehicle group exhibited a reduction in the number of c-Fos-positive cells in the CA1 area (F(3,20) = 6.301, p = 0.003 ISO+vehicle VS air+vehicle in multiple TSA injection improved CFC performance and hippocampal histone acetylation in mice that received repeated neonatal exposures to isoflurane. (A) TSA injection improved CFC performance in mice that received repeated neonatal exposures to isoflurane. The freezing times of each group (n = 12 mice per group) during CFC training and testing are shown. p<0.05. (B) Representative images of western blots showing histone acetylation levels in the CA1 hippocampal region for each group 1 h after CFC training. (C) Quantification of (B). p<0.05 versus the Air+vehicle group. Mean (SD) values are shown comparisons, p = 0.047 Fig 4B). TSA increased the number of c-Fos-positive cells in CA1 1 h after CFC training in mice with repeated neonatal exposure to isoflurane (ISO+vehicle VS ISO +TSA in multiple comparisons, p = 0.044, Fig 4B).The mechanism underlying neurocognitive impairment induced by inhalational anesthetic in the developing brain is thought to involve neurodegeneration, although the involved pathways are not entirely clear. The anesthetic properties of inhalational anesthetic are ascribed to a compound effect on the potentiation of -aminobutyric acid (GABAA)-gated receptors and inhibition of N-methyl-D-aspartate (NMDA)-gated receptors [31]. Several previous studies indicated that the ability of inhalational anesthetic to induce neurodegeneration in the developing brain is due to its effects on these two receptor types [324]. We performed repeated exposure to 0.75% isoflurane in neonatal mice and found that this mode of exposure can cause significant neurocognitive impairments without any changes in locomotor activity and anxiety levels in adult animals. Treatment with 0.75% isoflurane approximates an 0.5 minimum alveolar concentration (MAC) in adult mice, and the MAC in neonatal mice for isoflurane is higher than for adults [5]. In contrast to other studies describing that a single exposure with approximately 1 MAC concentration or prolonged exposure in the developing brain led to neurocognitive impairment, we found that the repeated neonatal exposure to a lower isoflurane dose could also induce neurocognitive impairment. We used CFC testing to assess mouse neurocognitive function this paradigm is commonly used to assess hippocampus-dependent associative learning and memory, and previous findings have demonstrated that the acetylation of several specific histone lysine residues (H3K9 14 and H4K581216) in the hippocampus might be related to CFC memory formation TSA injection improved c-Fos expression in response to CFC training in mice that received repeated neonatal exposures to isoflurane. (A) Representative immunohistochemistry images of c-Fos in the CA1 hippocampal region 1 h after CFC training. Hippocampal neuronal nuclei were stained purple with hematoxylin, and nuclear c-Fos protein is stained brown by DAB. (B) c-Fos-positive cell counts. Mice that received repeated neonatal exposures to isoflurane showed reduced numbers of c-Fos-positive neurons in the CA1 hippocampal region 1 h after CFC training, but this decrease was rescued by systemic TSA administration. p<0.05. Mean (SD) values are shown.We observed alterations in H3K9, H3K14, H4K5, and H4K12 acetylation in the CA1 hippocampal region at different time points after CFC training. Here, we choose to focus on the specific hippocampal subregion CA1 instead of whole hippocampus because CA1 is the major subreigion that has substantial reciprocal anatomical connections with the amygdala, which maybe hold significance for formation of CFC memory[37, 38],and several functional neuronal imaging studies demonstrated learning-related alterations occurred mainly in CA1 region instead of other subregions[391]. The results showed that the acetylation levels of these four histone residues were increased in the O2-enriched air exposure group 1 h after CFC training and returned to baseline levels within 24 h. In contrast to the O2-enriched air group, CFC training did not enhance H4K12 acetylation in the CA1 area of mice in the isoflurane exposure group. These data suggest that neurocognitive dysfunction induced by repeated exposure to isoflurane in neonatal mice was likely related to the dysregulation of hippocampal H4K12 acetylation. Histone acetylation is one of the mechanisms underlying local and global control of chromatin structure. A number of studies have reported that chromatin remodeling via histone acetylation responds quickly to various experiential stimuli to regulate downstream alterations in cellular gene expression [424]. Therefore, the dysregulation of histone acetylation following a learning task could result in deficiencies in memory-related gene expression and subsequent learning and memory dysfunction. In addition, it should be noted that the dysregulation of hippocampal histone acetylation only affected the specific lysine residue H4K12 in mice with neonatal isoflurane exposure. Peleg et al. [6] reported that impaired H4K12 acetylation also plays a role in age-related neurocognitive impairment because it leads to decreased expression of an array of genes underlying memory formation. It seems the neuropathologic basis for inhalational anesthetic-induced neurocognitive impairment may overlap with that for the cognitive dysfunction in the aged brain, although the shared underlying mechanisms remain unknown. Moreover, several previous studies[457] indicated that the acetylation of H3K9/14 and H4K5 also contributed greatly to learning, memory, and synaptic plasticity, and among them, the dysregulation of H3K9 acetylation has been reported to play a particularly important role in neurocognitive dysfunction related to iron overload2555206[7]. However, no changes in the acetylation of these lysine residues were detected in animals with memory impairment induced by neonatal isoflurane exposure. In previous studies, administration of global HDACis such as TSA or sodium butyrate was found to increase long-term potentiation (LTP) at Schaffer collaterals in CA1 of the hippocampus and rescue memory deficits in both aged and gene-mutant mice [6, 81]. Here, we found that TSA enhanced CFC memory formation in mice that underwent repeated neonatal exposure to isoflurane and exhibited memory impairment in adulthood but not in control mice. Similarly, several previous reports [8, 10, 48] also demonstrated that memory deficits in some genetic mouse models were mitigated by HDACi treatment, whereas normal cognitive function in wild-type mice was unaffected. Moreover, the negligible effect of TSA on CFC trials in the control group might be explained by the “ceiling effect” of animals’ fear response induced by an intense noxious stimulation. For instance, Itzhak et al. [48] reported that the maximum freezing response (“ceiling”) in mice during CFC trials was reached when the footshock current intensity reached 0.35 mA. It is possible that freezing responses are weakened and decrease to a lower level than the ceiling when the current intensity of footshock is less than 0.35 mA, in which case an effect of TSA on control mice with normal neurocognitive function could be detected. TSA is able to inhibit Class I (nuclear) and II (nuclear and cytoplasmic) HDACs both in vivo and in vitro, resulting in enhanced histone acetylation and subsequent changes in the expression of specific genes[49, 50]. We administered TSA (2 mg/kg) by intraperitoneal injection 2 h before CFC training, which was based on a previous study[8], and found that H3 and H4 acetylation in the CA1 hippocampal region markedly increased 1 h after CFC training.Notably, this model of TSA administration ameliorated the dysregulation of H4K12 acetylation following CFC training in mice with neonatal isoflurane exposure. We also observed changes of c-Fos expression 1 h after CFC training with or without TSA injection. c-Fos is considered a marker of synaptic plasticity and neuronal activation and is used to identify activated neurons for neuronal pathway tracing[51]. Our results showed that c-Fos expression was lower 1 h after CFC training in mice that received repeated neonatal isoflurane exposure, and the systemic TSA was able to rescue this gene expression deficit following the CFC task. These observations were in line with the observed effect on H4K12 acetylation. There is a wide range of mechanisms that contribute to the protective effect of HDACis on neurocognitive function, including epigenetic and non-epigenetic pathways [52, 53]. Most studies have focused on their blocking effects on HDACs, which increase the acetylation levels of histone and facilitated downstream gene products. In addition, several recent studies showed HDAC inhibitors improved the memory function also through the activation of specific genes mediated by the cAMP response element-binding protein (CREB)-CREB binding protein (CBP) transcriptional complex [36, 54, 55]. However, HDACis can also promote the acetylation of some non-histone substances that participate in learning and memory-related cellular and molecular processes. For instance, Green and coworkers[56] reported that nicotinamide’s inhibitory effect on class III NAD(+)-dependent HDACs rescued cognitive deficits in a transgenic mouse model of AD through a mechanism involving the increased acetylation alpha-tubulin and MAP2c and reduced Thr231-phospho-tau. Regardless of how many mechanisms remain to be identified, HDACis have shown greater therapeutic potential for cognitive defects caused by a variety of factors[6, 81].
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