BLA-involved circuits in neuropsychiatric disorders.

The basolateral amygdala (BLA) is the subregion of the amygdala located in the medial of the temporal lobe, which is connected with a wide range of brain regions to achieve diverse functions. Recently, an increasing number of studies have focused on the participation of the BLA in many neuropsychiatric disorders from the neural circuit perspective, aided by the rapid development of viral tracing methods and increasingly specific neural modulation technologies. However, how to translate this circuit-level preclinical intervention into clinical treatment using noninvasive or minor invasive manipulations to benefit patients struggling with neuropsychiatric disorders is still an inevitable question to be considered. In this review, we summarized the role of BLA-involved circuits in neuropsychiatric disorders including Alzheimer's disease, perioperative neurocognitive disorders, schizophrenia, anxiety disorders, depressive disorders, posttraumatic stress disorders, autism spectrum disorders, and pain-associative affective states and cognitive dysfunctions. Additionally, we provide insights into future directions and challenges for clinical translation.

Impaired synaptic plasticity and decreased glutamatergic neuron excitability induced by SIRT1/BDNF downregulation in the hippocampal CA1 region are involved in postoperative cognitive dysfunction.

BACKGROUND: Postoperative cognitive dysfunction (POCD) is a common complication after anesthesia/surgery, especially among elderly patients, and poses a significant threat to their postoperative quality of life and overall well-being. While it is widely accepted that elderly patients may experience POCD following anesthesia/surgery, the exact mechanism behind this phenomenon remains unclear. Several studies have indicated that the interaction between silent mating type information regulation 2 homologue 1 (SIRT1) and brain-derived neurotrophic factor (BDNF) is crucial in controlling cognitive function and is strongly linked to neurodegenerative disorders. Hence, this research aims to explore how SIRT1/BDNF impacts cognitive decline caused by anesthesia/surgery in aged mice. METHODS: Open field test (OFT) was used to determine whether anesthesia/surgery affected the motor ability of mice, while the postoperative cognitive function of 18 months old mice was evaluated with Novel object recognition test (NORT), Object location test (OLT) and Fear condition test (FC). The expressions of SIRT1 and other molecules were analyzed by western blot and immunofluorescence staining. The hippocampal synaptic plasticity was detected by Golgi staining and Long-term potentiation (LTP). The effects of SIRT1 and BDNF overexpression as well as chemogenetic activation of glutamatergic neurons in hippocampal CA1 region of 18 months old vesicular glutamate transporter 1 (VGLUT1) mice on POCD were further investigated. RESULTS: The research results revealed that older mice exhibited cognitive impairment following intramedullary fixation of tibial fracture. Additionally, a notable decrease in the expression of SIRT1/BDNF and neuronal excitability in hippocampal CA1 glutamatergic neurons was observed. By increasing levels of SIRT1/BDNF or enhancing glutamatergic neuron excitability in the CA1 region, it was possible to effectively mitigate synaptic plasticity impairment and ameliorate postoperative cognitive dysfunction. CONCLUSIONS: The decline in SIRT1/BDNF levels leading to changes in synaptic plasticity and neuronal excitability in older mice could be a significant factor contributing to cognitive impairment after anesthesia/surgery.

Repeated Ketamine Anesthesia during the Neonatal Period Impairs Hippocampal Neurogenesis and Long-Term Neurocognitive Function by Inhibiting Mfn2-Mediated Mitochondrial Fusion in Neural Stem Cells.

The mechanism of ketamine-induced neurotoxicity development remains elusive. Mitochondrial fusion/fission dynamics play a critical role in regulating neurogenesis. Therefore, this study was aimed to evaluate whether mitochondrial dynamics were involved in ketamine-induced impairment of neurogenesis in neonatal rats and long-term synaptic plasticity dysfunction. In the in vivo study, postnatal day 7 (PND-7) rats received intraperitoneal (i.p.) injection of 40 mg/kg ketamine for four consecutive times at 1 h intervals. The present findings revealed that ketamine induced mitochondrial fusion dysfunction in hippocampal neural stem cells (NSCs) by downregulating Mitofusin 2 (Mfn2) expression. In the in vitro study, ketamine treatment at 100 µM for 6 h significantly decreased the Mfn2 expression, and increased ROS generation, decreased mitochondrial membrane potential and ATP levels in cultured hippocampal NSCs. For the interventional study, lentivirus (LV) overexpressing Mfn2 (LV-Mfn2) or control LV vehicle was microinjected into the hippocampal dentate gyrus (DG) 4 days before ketamine administration. Targeted Mfn2 overexpression in the DG region could restore mitochondrial fusion in NSCs and reverse the inhibitory effect of ketamine on NSC proliferation and its faciliatory effect on neuronal differentiation. In addition, synaptic plasticity was evaluated by transmission electron microscopy, Golgi-Cox staining and long-term potentiation (LTP) recordings at 24 h after the end of the behavioral test. Preconditioning with LV-Mfn2 improved long-term cognitive dysfunction after repeated neonatal ketamine exposure by reversing the inhibitory effect of ketamine on synaptic plasticity in the hippocampal DG. The present findings demonstrated that Mfn2-mediated mitochondrial fusion dysfunction plays a critical role in the impairment of long-term neurocognitive function and synaptic plasticity caused by repeated neonatal ketamine exposure by interfering with hippocampal neurogenesis. Thus, Mfn2 might be a novel therapeutic target for the prevention of the developmental neurotoxicity of ketamine.

Dynorphin promotes stress-induced depressive behaviors by inhibiting ventral pallidal neurons in rats.

AIM: Endogenous dynorphin signaling via kappa opioid receptors (KORs) plays a key role in producing the depressive and aversive consequences of stress. We investigated the behavioral effects of the dynorphin/KOR system in the ventral pallidum (VP) and studied the underlying mechanisms. METHODS: To investigate the effects of dynorphin on the VP, we conducted behavioral experiments after microinjection of drugs or shRNA and brain-slice electrophysiological recordings. Histological tracing and molecular biological experiments were used to identify the distribution of KORs and the possible sources of dynorphin projections to the VP. RESULTS: An elevated dynorphin concentration and increased KOR activity were observed in the VP after acute stress. Infusion of dynorphin-A into the VP produced depressive-like phenotypes including anhedonia and despair and anxiety behaviors, but did not alter locomotor behavior. Mechanistically, dynorphin had an inhibitory effect on VP neurons-reducing their firing rate and inhibiting excitatory transmission-through direct activation of KORs and modulation of downstream G-protein-gated inwardly rectifying potassium (GIRK) channels and high-voltage gated calcium channels (VGCCs). Tracing revealed direct innervation of VP neurons by dynorphin-positive projections; potential sources of these dynorphinergic projections include the nucleus accumbens, amygdala, and hypothalamus. Blockade of dynorphin/KOR signaling in the VP by drugs or viral knock-down of KORs significantly reduced despair behavior in rats. CONCLUSIONS: Endogenous dynorphinergic modulation of the VP plays a critical role in mediating depressive reactions to stress.

The Role of Epigenetic Modifications in Neurotoxicity Induced by Neonatal General Anesthesia.

It has been widely demonstrated by numerous preclinical studies and clinical trials that the neonates receiving repeated or long-time general anesthesia (GA) could develop prolonged cognitive dysfunction. However, the definite mechanism remains largely unknown. Epigenetics, which is defined as heritable alterations in gene expression that are not a result of alteration of DNA sequence, includes DNA methylation, histone post-translational modifications, non-coding RNAs (ncRNAs), and RNA methylation. In recent years, the role of epigenetic modifications in neonatal GA-induced neurotoxicity has been widely explored and reported. In this review, we discuss and conclude the epigenetic mechanisms involving in the process of neonatal anesthesia-induced cognitive dysfunction. Also, we analyze the wide prospects of epigenetics in this field and its possibility to work as treatment target.

Neonatal Exposure to Propofol Interferes with the Proliferation and Differentiation of Hippocampal Neural Stem Cells and the Neurocognitive Function of Rats in Adulthood via the Akt/p27 Signaling Pathway.

Objective: Neonatal exposure to propofol has been reported to cause neurotoxicity and neurocognitive decline in adulthood; however, the underlying mechanism has not been established. Methods: SD rats were exposed to propofol on postnatal day 7 (PND-7). Double-immunofluorescence staining was used to assess neurogenesis in the hippocampal dentate gyrus (DG). The expression of p-Akt and p27 were measured by western blotting. The Morris water maze, novel object recognition test, and object location test were used to evaluate neurocognitive function 2-month-old rats. Results: Phosphorylation of Akt was inhibited, while p27 expression was enhanced after neonatal exposure to propofol. Propofol also inhibited proliferation of neural stem cells (NSCs) and decreased differentiation to neurons and astroglia. Moreover, the neurocognitive function in 2-month-old rats was weakened. Of significance, intra-hippocampal injection of the Akt activator, SC79, attenuated the inhibition of p-AKT and increase of p27 expression. SC79 also rescued the propofol-induced inhibition of NSC proliferation and differentiation. The propofol-induced neurocognition deficit was also partially reversed by SC79. Conclusion: Taken together, these results suggest that neurogenesis is hindered by neonatal propofol exposure. Specifically, neonatal propofol exposure was shown to suppress the proliferation and differentiation of NSCs by inhibiting Akt/p27 signaling pathway.

The Effect of SIRT3/Ac-SOD2 Mediated Oxidative Stress and HCN1 Channel Activity on Anesthesia/Surgery Induced Anxiety-Like Behavior in Mice.

Anxiety disorders are the most common psychiatric diseases, and perioperative factors often increase the incidence of anxiety. However, the mechanism and treatment for perioperative anxiety, especially anesthesia/surgery-induced postoperative anxiety, are largely unknown. Sirtuin 3 (SIRT3) which located in the mitochondria is the NAD-dependent deacetylase protein. SIRT3 mediated oxidative stress is associated with several neuropsychiatric diseases. In addition, hyperpolarization-activated cyclic nucleotide-gated 1 (HCN1) channel is also reported involved in anxiety symptoms. The purpose was to assess the role of SIRT3 on postoperative anxiety like behavior in C57/BL6 mice. We found that SIRT3 level reduced and HCN1 expression level increased in mice medial prefrontal cortex (mPFC) as well as anxiety like behavior postoperatively. In interventional research, SIRT3 adeno-associated virus vector or control vector was injected into the mPFC brain region. Enzyme-linked immunosorbent assay, immunofluorescence staining, and western blotting were employed to detect oxidative stress reactions and HCN1 channel activity. SIRT3 overexpression attenuated postoperative anxiety in mice. Superoxide dismutase 2 (SOD2) acetylation levels, SOD2 oxidative stress activity, mitochondrial membrane potential levels, and HCN1 channels were also inhibited by SIRT3 overexpression. Furthermore, the HCN1 channel inhibitor ZD7288 significantly protected against anesthesia/surgery-induced anxiety, but without SIRT3/ac-SOD2 expression or oxidative stress changes. Our results suggest that SIRT3 may achieve antianxiety effects through regulation of SOD2 acetylation-mediated oxidative stress and HCN1 channels in the mPFC, further strengthening the therapeutic potential of targeting SIRT3 for anesthesia/surgery-induced anxiety-like behavior.

Hippocampal SIRT1-Mediated Synaptic Plasticity and Glutamatergic Neuronal Excitability Are Involved in Prolonged Cognitive Dysfunction of Neonatal Rats Exposed to Propofol.

Neonates who receive repeated or prolonged general anesthesia before the age of 4 are at a significantly higher risk of developing cognitive dysfunction later in life. In this study, we investigated the effects of repeated neonatal propofol exposure on hippocampal synaptic plasticity, neuronal excitability, and cognitive function. Adeno-associated SIRT1 virus with CaMKIIɑ promotor and a viral vector carrying the photosensitive gene ChR2 with the CaMKIIɑ promotor, as well as their control vectors, were stereotaxically injected into the hippocampal CA1 region of postnatal day 5 (PND-5) rats. PND-7 rats were given intraperitoneal injection of 60 mg/kg propofol or fat emulsion for three consecutive days. Western blotting, Golgi staining, and double immunofluorescence staining were used to evaluate the SIRT1 expression, synaptic plasticity, and the excitability of neurons in the hippocampal CA1 region. The Morris water maze (MWM) test was conducted on PND-30 to assess the learning and memory abilities of rats. Repeated neonatal propofol exposure reduced SIRT1 expression, suppressed synaptic plasticity, decreased glutamatergic neuron excitability in the hippocampus, and damaged learning and memory abilities. Overexpression of SIRT1 attenuated propofol-induced cognitive dysfunction, excitation-inhibition imbalance, and synaptic plasticity damage. After optogenetic stimulation of glutamatergic neurons in the hippocampal CA1 region, the learning and memory abilities of rats exposed to propofol were improved on PND-30. Our findings demonstrate that SIRT1 plays an important role in cognitive dysfunction induced by repeated neonatal propofol exposure by suppressing synaptic plasticity and neuronal excitability.

Neonatal Anesthesia by Ketamine in Neonatal Rats Inhibits the Proliferation and Differentiation of Hippocampal Neural Stem Cells and Decreases Neurocognitive Function in Adulthood via Inhibition of the Notch1 Signaling Pathway.

The Notch signaling pathway plays an important role in the regulation of neurogenesis. The objective of this study was to investigate whether the Notch signaling pathway was involved in the neurogenesis impairment and long-term neurocognitive dysfunction caused by neonatal exposure to ketamine. On postnatal day 7 (PND-7), male Sprague-Dawley (SD) rats were intraperitoneally injected with 40 mg/kg ketamine four consecutive times (40 mg/kg × 4) at 1-h intervals. Notch ligand Jagged1 (0.5 mg/kg) and lentivirus overexpressing the Notch1 intracellular domain (LV-NICD1) were microinjected into the hippocampal dentate gyrus (DG) 1 h or 4 days before ketamine administration, respectively. The expression of Notch1 signaling pathway-related proteins was detected by Western blotting 24 h after ketamine administration. The proliferation and differentiation of the neural stem cells (NSCs) in the hippocampal DG were evaluated by double immunofluorescence staining 24 h after treatment. Moreover, changes in hippocampus-dependent spatial memory of 2-month-old rats were investigated with the Morris water maze test. Ketamine anesthesia in neonatal rats decreased the expression levels of Jagged1, Notch1, NICD1, and hairy enhancer of split 1 (Hes1); inhibited the proliferation and astrocytic differentiation of NSCs; and promoted the differentiation of neurons. Neonatal exposure to ketamine caused deficits in hippocampus-dependent spatial reference memory tasks in 2-month-old rats. Microinjection of Jagged1 or LV-NICD1 reversed the inhibitory effect of ketamine on the expression of Notch1-related proteins in the hippocampal DG, attenuated the ketamine-mediated decrease in NSC proliferation and differentiation, and improved the cognitive function of 2-month-old rats after neonatal exposure to ketamine. These results suggest that neonatal exposure to ketamine in rats inhibits the proliferation and differentiation of hippocampal NSCs and impairs neurocognitive function in adulthood. The Notch1 signaling pathway may be involved in the impairment of hippocampus-dependent learning and memory during adulthood caused by neonatal exposure to ketamine. These findings contribute to further understanding the neurotoxicity induced by neonatal exposure to ketamine and the underlying mechanisms.

Downregulated SIRT1 in the CeA is involved in chronic pain-depression comorbidity.

Comorbid chronic pain and depression are increasingly becoming a concerning public problem, but the underlying mechanisms remain unclear. Here, we demonstrate that pain-related depression-like behaviors are induced in a rat model of chronic constriction injury (CCI). Using this model, we found that chronic neuropathic pain decreased the activity and expression of sirtuin 1 (SIRT1, an NAD+-dependent deacetylase) in the central nucleus of the amygdala (CeA). In addition, the pharmacologic activation of SIRT1 in the CeA could alleviate the depression-like behaviors associated with chronic pain while relieving sensory pain. Accordingly, adeno-associated virus (AAV)-mediated SIRT1 overexpression in the CeA produced a positive effect on the easement of chronic pain and comorbid depression. Taken together, these findings highlight the role of SIRT1 in the CeA in chronic pain and depression states and reveal that the upregulation of SIRT1 may be a potential therapy for the treatment of pain-depression comorbidities.

Ca2+-PKCα-ERK1/2 signaling pathway is involved in the suppressive effect of propofol on proliferation of neural stem cells from the neonatal rat hippocampus.

Neonatal exposure to propofol induces persistent behavioral abnormalities in adulthood. In addition to triggering the apoptosis of neurons in the developing brain, anesthetics may contribute to the development of cognitive deficits by interfering neurogenesis. Given the importance of neural stem cell (NSC) proliferation in neurogenesis, the effect of propofol on NSC proliferation and the mechanisms underlying this effect were investigated. Hippocampal NSC proliferation from neonatal rats was examined using 5-bromo-2'-deoxyuridine incorporation assays in vitro. The [Ca2+]i was analyzed using flow cytometry. The activations of protein kinase C (PKC)-α and extracellular signal-regulated kinases 1/2 (ERK1/2) were measured by western blot. Our results showed that propofol significantly inhibited NSC proliferation in vitro. [Ca2+]i and activations of PKCα and ERK1/2 in NSCs were markedly suppressed by propofol (5, 10, 20, 40 and 80 µM). Ca2+ channel blocker verapamil, PKCα inhibitor chelerythrine and ERK1/2 kinase inhibitor PD98059 exerted their maximal effects on NSC function at concentrations of 20, 10 and 20 µM, respectively. Propofol (20 µM) could not produce further additional suppression effects when used in combination with verapamil (20 µM), chelerythrine (10 µM) or PD98059 (20 µM). In addition, phorbol-12-myristate-13-acetate (PMA, a activator of PKC) markedly attenuated the suppressive effects of propofol on ERK1/2 phosphorylation and NSC proliferation. The inhibition effects on PKCα activation, ERK1/2 phosphorylation and NSC proliferation induced by propofol were significantly improved by BayK8644 (a calcium channel agonist). These results indicate that propofol can inhibits hippocampal NSC proliferation by suppressing the Ca2+-PKCα-ERK1/2 signaling pathway.

Potential roles of insect Tropomyosin1-X1 isoform in the process of Candidatus Liberibacter asiaticus infection of Diaphorina citri.

The Asian citrus psyllid (ACP), Diaphorina citri Kuwayama, is the transmitting vector of Candidatus Liberibacter asiaticus (CLas), which causes citrus disease Huanglongbing (HLB). In recent years, control of HLB has been achieved by reducing the vector population. In the present study, we identified an isoform of D. citri tropomyosin (herein designated as DcTm1-X1). DcTm1-X1 was down-regulated in CLas-infected ACPs compared with uninfected ACPs. Bioinformatics analysis revealed that the full-length DcTm1-X1 is 2955 bp and encodes a protein of 284 amino acids with a deduced molecular weight of 32.15 kDa. Phylogenetic tree analysis suggested that DcTm1-X1 shares a high amino acid identity with its homolog in Acyrthosiphon pisum. Higher DcTm1-X1 expression levels were found in the leg of the psyllid by reverse transcription quantitative PCR (RT-qPCR). According to Blue Native PAGE analysis and mass spectrometric analysis, DcTm1-X1 interacts with citrate synthase (CS) and V-type proton ATPase subunit B-like (VAT). In addition, knockdown of DcTm1-X1 by RNA interference (RNAi) significantly increased the mortality rate of nymphs and the infection rate of CLas at different time points. Taken together, our results show that DcTm1-X1 might play an important role in response to CLas, but also lay a foundation for further research on the functions of DcTm1-X1.

SIRT1 attenuates neuropathic pain by epigenetic regulation of mGluR1/5 expressions in type 2 diabetic rats.

Accumulating evidence has demonstrated that epigenetic modification-mediated changes in pain-related gene expressions play an important role in the development and maintenance of neuropathic pain. Sirtuin 1 (SIRT1), a nicotinamide adenine dinucleotide (NAD)-dependent deacetylase, is involved in the development of chronic pain. Moreover, SIRT1 may be a novel therapeutic target for the prevention of type 2 diabetes mellitus (T2DM). But the role of SIRT1 in T2DM-induced neuropathic pain remains unknown. In this study, we found that spinal SIRT1 expression and activity were downregulated significantly in high-fat-fed/low-dose streptozotocin-induced neuropathic pain rats. SIRT1 localized in spinal neurons but not in astrocytes or microglia. Furthermore, the expressions of metabotropic glutamate receptor (mGluR1) and mGluR5, which play a key role in central sensitization and neuropathic pain, and H3 acetylation levels at Grm1/5 (encoding mGluR1/5) promoter regions were increased in diabetic neuropathic pain rats. SIRT1 activator SRT1720 reversed thermal hyperalgesia and mechanical allodynia and spinal neuronal activation in diabetic neuropathic pain rats. Concurrently, increased expressions of mGluR1/5 and H3 acetylation levels at Grm1/5 promoter regions were reversed by SIRT1 activation. In addition, knockdown of SIRT1 by Ad-SIRT1-shRNA induced pain behaviors and spinal neuronal activation in normal rats, which was accompanied by the increased expressions of mGluR1/5 and H3 acetylation levels at Grm1/5 promoter regions. Therefore, we concluded that SIRT1-mediated epigenetic regulation of mGluR1/5 expressions was involved in the development of neuropathic pain in type 2 diabetic rats.

Urotensin II inhibitor eases neuropathic pain by suppressing the JNK/NF-κB pathway.

Urotensin II (U-II), a cyclic peptide originally isolated from the caudal neurosecretory system of fishes, can produce proinflammatory effects through its specific G protein-coupled receptor, GPR14. Neuropathic pain, a devastating disease, is related to excessive inflammation in the spinal dorsal horn. However, the relationship between U-II and neuropathic pain has not been reported. This study was designed to investigate the effect of U-II antagonist on neuropathic pain and to understand the associated mechanisms. We reported that U-II and its receptor GPR14 were persistently upregulated and activated in the dorsal horn of L4-6 spinal cord segments after chronic constriction injury (CCI) in rats. Intrathecal injection of SB657510, a specific antagonist against U-II, reversed CCI-induced thermal hyperalgesia and mechanical allodynia. Furthermore, we found that SB657510 reduced the expression of phosphorylated c-Jun N-terminal kinase (p-JNK) and nuclear factor-κB (NF-κB) p65 as well as subsequent secretion of interleukin-1ß (IL-1ß), IL-6 and tumor necrosis factor-α (TNF-α). It was also showed that both the JNK inhibitor SP600125 and the NF-κB inhibitor PDTC significantly attenuated thermal hyperalgesia and mechanical allodynia in CCI rats. Our present research showed that U-II receptor antagonist alleviated neuropathic pain possibly through the suppression of the JNK/NF-κB pathway in CCI rats, which will contribute to the better understanding of function of U-II and pathogenesis of neuropathic pain.

Propofol Inhibits Lipopolysaccharide-Induced Inflammatory Responses in Spinal Astrocytes via the Toll-Like Receptor 4/MyD88-Dependent Nuclear Factor-κB, Extracellular Signal-Regulated Protein Kinases1/2, and p38 Mitogen-Activated Protein Kinase Pathways.

BACKGROUND: In this study, we investigated the effect of propofol, a commonly used IV anesthetic, on lipopolysaccharide (LPS)-induced inflammatory responses in astrocytes and explored the molecular mechanisms by which it occurs. METHODS: Astrocytes were stimulated with LPS (1.0 µg/mL) in the absence and presence of different concentrations of propofol. The expression of astrocyte marker glial fibrillary acidic protein (GFAP) in astrocytes was detected using immunofluorescence staining and Western blot analysis. The levels of interleukin (IL)-1ß, IL-6, and tumor necrosis factor-α were measured using an enzyme-linked immunosorbent assay. The mRNA level of Toll-like receptor 4 (TLR4) was determined by semiquantitative reverse transcriptase-polymerase chain reaction. The protein expressions of TLR4, myeloid differentiation factor 88 (MyD88), p- extracellular signal-regulated protein kinases (ERK)1/2, p-c-Jun N-terminal kinase, p-p38 mitogen-activated protein kinase (MAPK), p-I-κBα, I-κBα, and p-nuclear factor-κB (NF-κB)p65 were detected by Western blot. RESULTS: Our results show that after stimulation with LPS, the levels of IL-1ß, IL-6, and tumor necrosis factor-α and the expression of GFAP in astrocytes were up-regulated significantly. In addition, the expression of TLR4, MyD88, p-ERK1/2, p-c-Jun N-terminal kinase, p-p38 MAPK, and p-NF-κBp65 increased, whereas the expression of total I-κBα decreased upon stimulation with LPS. Propofol (10 µM) reduced the secretion of proinflammatory cytokines, inhibited the expressions of GFAP, TLR4, MyD88, p-ERK1/2, p-p38 MAPK, and p-NF-κBp65 in astrocytes challenged with LPS. CONCLUSIONS: In the present study, propofol 10 µM but not lower clinically relevant or higher supra-clinical concentrations attenuated LPS-induced astrocyte activation and subsequent inflammatory responses by inhibiting the TLR4/MyD88-dependent NF-κB, ERK1/2, and p38 MAPK pathways.

Ketamine interferes with the proliferation and differentiation of neural stem cells in the subventricular zone of neonatal rats.

BACKGROUND: Previous studies have shown ketamine can alter the proliferation and differentiation of neural stem cells (NSCs) in vitro. However, these effects have not been entirely clarified in vivo in the subventricular zone (SVZ) of neonatal rats. The present study was designed to investigate the effects of ketamine on the proliferation and differentiation of NSCs in the SVZ of neonatal rats in vivo. METHODS: Postnatal day 7 (PND-7) male Sprague-Dawley rats were administered four injections of 40 mg/kg ketamine at 1-h intervals, and then 5-bromodeoxyuridine (BrdU) was injected intraperitoneally at PND-7, 9 and 13. NSC proliferation was assessed with Nestin/BrdU double-labeling immunostaining. Neuronal and astrocytic differentiation was evaluated with ß-tubulin III/BrdU and GFAP/BrdU double-labeling immunostaining, respectively. The expressions of nestin, ß-tubulin III and GFAP were measured using Western blot analysis. The apoptosis of NSCs and astrocytes in the SVZ of neonatal rats was evaluated using nestin/caspase-3 and GFAP/caspase-3 double-labeling immunostaining. RESULTS: Neonatal ketamine exposure significantly reduced the number of nestin/BrdU and GFAP/BrdU double-positive cells in the SVZ. Meanwhile, the expressions of nestin and GFAP in the SVZ from the ketamine group were significantly decreased compared those in the control group. Still, no double-positive cells for nestin/caspase-3 and GFAP/caspase-3 were found after ketamine exposure. In addition, the neuronal differentiation of NSCs in the SVZ was markedly promoted by ketamine with an increased number of ß-tubulin III/BrdU double-positive cells and enhanced expression of ß-tubulin III. These effects of ketamine on the NSCs in the SVZ often lasted at least 1 week after ketamine anesthesia. CONCLUSION: In the present study, it was demonstrated that ketamine could alter neurogenesis by inhibiting the proliferation of NSCs, suppressing their differentiation into astrocytes and promoting the neuronal differentiation of the NSCs in the SVZ of neonatal rats during a critical period of their neurodevelopment.

Ketamine inhibits proliferation of neural stem cell from neonatal rat hippocampus in vitro.

BACKGROUND/AIMS: Ketamine is a widely used anesthetic in obstetric and pediatric anesthesia. In the developing brain, the widespread neuron apoptosis triggered by ketamine has been demonstrated. However, little is known about its effect on neural stem cells (NSCs) function. This study aimed to investigate the effect of ketamine on proliferation of NSCs from neonatal rat hippocampus. METHODS: Neural stem cells were isolated from the hippocampus of Sprague-Dawley rats on postnatal day 3. In dose-response experiments, cultured neural stem cells (NSCs) were exposed to different concentrations of ketamine (0-1000 µM) for 24 hrs. The proliferative activity of NSCs was evaluated by 5-Bromo-2'-deoxyuridine (BrdU) incorporation assay. Apoptosis of neural stem cells were assessed using caspase-3 by western blot. The intracellular Ca(2+) concentration ([Ca(2+)]i) in NSCs was analyzed by flow cytometry. The activation of protein kinase C-α (PKCα) and the phosphorylation of extracellular signal-regulated kinases 1/2 (ERK1/2) were measured by western blot analysis. RESULTS: Clinical relevant concentration of ketamine (10, 20 and 50 µM) did not markedly alter the proliferation of NSCs from neonatal rat hippocampus in vitro. However, ketamine (200, 500, 800 and 1000µM) significantly inhibited the proliferation of NSCs and did not affect the expression of caspase-3. Meanwhile, ketamine (200, 500, 800 and 1000µM) also markedly decreased [Ca(2+)]i as well as suppressed PKCα activation and ERK1/2 phosphorylation in NSCs. A combination of subthreshold concentrations of ketamine (100 µM) and Ca(2+) channel blocker verapamil (2.5 µM), PKCα inhibitor chelerythrine (2.5 µM) or ERK1/2 kinase inhibitor PD98059 (5 µM) significantly produced suprathreshold effects on PKCα activation, ERK1/2 phosphorylation and NSC proliferation. CONCLUSION: Ketamine inhibited proliferation of NSCs from neonatal rat hippocampus in vitro. Suppressing Ca(2+)-PKCα-ERK1/2 signaling pathway may be involved in this inhibitory effect of ketamine on NSCs proliferation.

Salusin-ß, but not salusin-α, promotes human umbilical vein endothelial cell inflammation via the p38 MAPK/JNK-NF-κB pathway.

OBJECTIVE: Recently, salusin-ß has been reported to have pro-atherosclerotic effects, but salusin-α has anti-atherosclerotic effects. Our previous study has shown that salusin-ß but not salusin-α promotes vascular inflammation in apoE-deficient mice. However, the underlying mechanism remains unknown. In this study, we observed the effect of salusins on inflammatory responses and the MAPK-NF-κB signaling pathway in human umbilical vein endothelial cells (HUVECs). METHODS AND RESULTS: HUVECs were incubated with different concentrations of salusin-α and salusin-ß. The levels of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were determined using enzyme-linked immunosorbent assay (ELISA). The mRNA expressions of vascular cell adhesion molecule-1 (VCAM-1) and monocyte chemoattractant protein-1 (MCP-1) were quantified using quantitative real-time polymerase chain reaction (PCR). The protein expressions of VCAM-1, MCP-1, I-κBα, NF-κB, p-JNK and p-p38 MAPK were measured using western blotting analysis. Our results showed that in HUVECs, salusin-ß could up-regulate the levels of IL-6, TNF-α, VCAM-1 and MCP-1, promote I-κBα degradation and NF-κB activation, and increase the phosphorylation of JNK and p38 MAPK. These effects could be inhibited by p38 MAPK inhibitor SB203580 and/or JNK inhibitor SP600125. In contrast, salusin-α could selectively decrease VCAM-1 protein, but did not show any effect on the expressions of VCAM-1 mRNA, TNF-α, IL-6, MCP-1, I-κBα, NF-κB, p-JNK or p-p38 MAPK. CONCLUSION: Salusin-ß was able to promote inflammatory responses in HUVECs via the p38 MAPK-NF-κB and JNK-NF-κB pathways. In contrast, salusin-α failed to show any significant effects on the inflammatory responses in HUVECs. These results provide further insight into the mechanisms behind salusins in vascular inflammation and offer a potential target for the prevention and treatment of atherosclerosis.

Salusin-ß not salusin-α promotes vascular inflammation in ApoE-deficient mice via the I-κBα/NF-κB pathway.

OBJECTIVE: Vascular inflammation plays an important role in the development and progression of atherosclerosis. Recently, salusins (salusin-α and salusin-ß) have been reported to be associated wtih atherosclerosis. However, its underlying mechanism remains incompletely known. In this study, we observed the effects of salusins on vascular inflammation in apoE-deficient (apoE-/-) mice. METHODS AND RESULTS: Six-week old male apoE-/- mice were infused with salusin-α, salusin-ß or vehicle for 8 weeks via osmotic mini-pumps. Our results showed that apoE-/- mice receiving vehicle alone developed severe atherosclerotic lesions and dyslipidemia, with significantly up-regulated levels of IL-6, TNF-α, VCAM-1 and MCP-1. For apoE-/- mice receiving 8 weeks of salusin-ß infusion, the atherosclerotic lesions were markedly aggravated, and the levels of IL-6, TNF-α, VCAM-1 and MCP-1 were substantially increased, despite a similar plasma lipid concentration with that of apoE-/- mice. However, after 8 week-infusion of salusin-α, apoE-/- mice presented significant amelioration in atherosclerotic lesions, along with remarkably up-regulated level of high-density lipoprotein-cholesterol (HDL-C) and down-regulated levels of IL-6 and TNF-α, but without any effect on the expressions of VCAM-1 and MCP-1. Furthermore, the activation of nuclear factor-κB (NF-κB), an important transcription factor essential for inflammatory molecules, and the degradation of I-κBα, an inhibitor of NF-κB, were markedly increased in apoE-/- mice receiving vehicle alone. Treatment with salusin-ß not salusin-α could remarkably accelerate the process of NF-κB nuclear translocation and I-κBα degradation. CONCLUSION: Salusin-ß, but not salusin-α, promotes vascular inflammation in apoE-deficient mice via the I-κBα/NF-κB pathway. These findings provide further insight into the mechanism of salusins in atherosclerosis and potential targets for the prevention and treatment of atherosclerosis.

Ghrelin alleviates neuropathic pain through GHSR-1a-mediated suppression of the p38 MAPK/NF-κB pathway in a rat chronic constriction injury model.

BACKGROUND AND OBJECTIVES: Neuropathic pain is related to the sustained activation of neuroglial cells and the production of proinflammatory cytokines in the spinal dorsal horn. Ghrelin, the endogenous ligand for growth hormone secretagogue receptor 1a (GHSR-1a), has been shown to inhibit the activation of microglia and the release of proinflammatory cytokines. The purpose of this study was to investigate the role of ghrelin/GHSR-1a signaling in neuropathic pain and to understand the associated mechanisms. METHODS: A rat model of neuropathic pain was established by chronic constriction injury (CCI) of the sciatic nerve. Hyperalgesia and allodynia were evaluated by observing the mechanical withdrawal threshold and the thermal withdrawal latency. The expression levels of ghrelin and GHSR-1a were detected by semiquantitative reverse transcriptase-polymerase chain reaction and Western blot analysis. The levels of interleukin-1ß (IL-1ß), IL-6, and tumor necrosis factor-α were detected using an enzyme-linked immunosorbent assay. The expression levels of p-p38 mitogen-activated protein kinases (p38 MAPK) and nuclear factor-κB (NF-κB) p65 were determined by Western blot and immunohistochemistry analysis. RESULTS: Both ghrelin and GHSR-1a were expressed in the spinal dorsal horns of normal rats and were not significantly different from that of sham rats. However, rats in the CCI model group developed severe hyperalgesia and allodynia, as well as significantly downregulated expression of ghrelin and GHSR-1a. Compared with CCI model rats, intrathecal injection of ghrelin clearly delayed thermal hyperalgesia and mechanical allodynia at 3, 5, and 7 days after CCI; reduced the levels of IL-1ß, IL-6, and tumor necrosis factor-α; and inhibited the phosphorylation of p38 MAPK and the activation of NF-κBp65 in the spinal dorsal horn. In addition, the effect of ghrelin could be blocked by [D-Lys]-GHRP-6, a GHSR-1a antagonist. CONCLUSIONS: Our present study demonstrated that ghrelin alleviated neuropathic pain through a GHSR-1a-mediated suppression of the p38 MAPK/NF-κB pathway.