Prolonged sevoflurane exposure causes abnormal synapse development and dysregulates beta-neurexin and neuroligins in the hippocampus in neonatal rats.

BACKGROUND: The underlying molecular mechanisms of the excitatory/inhibitory (E/I) imbalance induced by sevoflurane exposure to neonates remain poorly understood. This study aimed to investigate the long-term effects of prolonged sevoflurane exposure to neonatal rats during the peak period of synaptogenesis on the changes of trans-synaptic neurexin-neuroligin interactions, synaptic ultrastructure in the hippocampus and cognition. METHODS: A total of 30 rat pups at postnatal day (P) 7 was randomly divided into two groups: the control group (exposed to 30 % oxygen balanced with nitrogen) and the sevoflurane group (exposed to 2.5 % sevoflurane plus 30 % oxygen balanced with nitrogen) for 6 h. Neurocognitive behaviors were assessed with the Open field test at P23-25 and the Morris water maze test at P26-30. The expression of ß-neurexin (ß-NRX), N-methyl-d-aspartate receptor 2 subunit (NR2A and NR2B), neuroligin-1 (NLG-1), neuroligin-2 (NLG-2), postsynaptic density protein-95 (PSD-95), α1-subunit of the γ-aminobutyric acid A receptor (GABAAα1) and gephyrin in the hippocampus at P30 were measured by Western blot. The ultrastructure of synapses was examined under electron microscope. RESULTS: Prolonged sevoflurane exposure at P7 resulted in cognitive deficiency in adolescence, as well as the downregulation of ß-NRX, NR2A, NR2B, NLG-1, and PSD-95, and the upregulation of GABAAα1, NLG-2, and gephyrin in the hippocampal CA3 region. Sevoflurane anesthesia also increased the number of symmetric synapses in the hippocampus. CONCLUSIONS: Prolonged sevoflurane exposure during the brain development leads to cognitive deficiency and disproportion of excitatory/inhibitory synapses which may be caused by dysregulated expression of synaptic adhesion molecules of ß-NRX and neuroligins.

Metformin suppresses lung adenocarcinoma by downregulating long non-coding RNA (lncRNA) AFAP1-AS1 and secreted phosphoprotein 1 (SPP1) while upregulating miR-3163.

AFAP1-AS1 plays a pro-tumor role in lung cancer. However, no investigation has focused on whether it is involved in the anticancer activity of metformin (Met) in the treatment of lung adenocarcinoma (LUAD). Reverse transcription quantitative polymerase chain reaction (RT-qPCR) was performed to detect the expression of long non-coding (lnc)RNA AFAP1-AS1, the microRNA (miR)-3163, and secreted phosphoprotein 1 (SPP1) in LUAD tissues, or of A549 and H3122 cells. Cell Counting Kit-8, wound scratch, and cell invasion assays were performed to evaluate the effect of the overexpression of lncRNA AFAP1-AS1, miR-3163, and SPP1 on the malignant behaviors of A549 and H3122 cells. Phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR) signaling pathway-related proteins were detected by Western blot analysis. Dual luciferase reporter or RIP assays were used to determine the interplay between AFAP1-AS1 and miR-3163, or of miR-3163 and SPP1. Met inhibits the malignant characteristics of A549 and H3122 cells in vitro. GEPIA database analysis showed that AFAP1-AS1 is a highly expressed lncRNA in LUAD tissues, which was validated by RT-qPCR. Overexpression of AFAP1-AS1 suppressed the met-mediated anti-tumor activity in A549 and H3122 cells, while AFAP1-AS1 silencing promoted it. Met inhibited AFAP1-AS1 expression, which resulted in reduced proliferation, migration, and invasion in A549 and H3122 cells. This led to AFAP1-AS1-mediated suppression of miR-3163 and, subsequently, the upregulation of SPP1. Met exerts its antitumor activities by regulating the AFAP1-AS1/miR-3163/SPP1/PI3K/Akt/mTOR axis. Our findings deepen our understanding of mechanisms underlying anti-tumor effect of Met in LUAD.

Flexible and simple telecentric camera calibration method.

The measurement accuracy of telecentric imaging technique straightforwardly depends on the calibration of the telecentric camera. We present a flexible and simple calibration method based on a telecentric imaging model and orthogonality of rotation matrix. First, we use the orthogonality of rotation matrix to solve the magnification. Second, the external parameters are solved by the imaging model, and the ambiguity of sign is solved. Finally, we use the LM nonlinear optimization method to solve the distortion parameters. Experimental results show that the reprojection error is 0.7 pixels, which represents the actual dimension of 6.37 µm. In addition, the standard measuring block and real objects are measured, and the results verify the measurement accuracy and robustness of the proposed method.

Triiodothyronine attenuates neurocognitive dysfunction induced by sevoflurane in the developing brain of neonatal rats.

BACKGROUND: Whilst concerns have been raised about the detrimental effects of general anaesthetics on the brain's development and function in the young, reports have indicated that thyroid hormones are able to promote neurogenesis in the developing brain. This present study aimed to investigate the effects of triiodothyronine (T3) on the neonatal rat brain, following sevoflurane exposure. METHODS: Postnatal day 7 (P7) ratpups were treated with Triiodothyronine (T3) (1 µg/100 g body weight, i.p. injection, once/day for 3 days) after 2% sevoflurane exposure for 6 h. They were sacrificed at either P7 (immediately), P15 or P30 and their brains were harvested to assess cell death, proliferation in the hippocampus, N-methyl-D-aspartate (NMDA) receptor subunit A and B, and a post-synaptic protein (PSD-95 in the hippocampus,). Neuro-behavioral changes in other cohorts between P27 and P30 were evaluated with Morris water maze and open field tests. RESULTS: Sevoflurane exposure caused cell death and suppressed the proliferation of astrocytes and neurons, as well as the dendritic growth of neurons in the hippocampus which were all reversed by the administration of T3. Moreover, cognitive function, including learning, memory, and adaptability to a new environment, were impaired by sevoflurane exposure, which was also negated by T3 treatment. Furthermore, sevoflurane decreased the expression of NMDA receptor subunits NR2A and NR2B, as well as PSD-95 in the hippocampus at P15 and those effects of sevoflurane were abolished by T3 administration. CONCLUSIONS: A potential therapeutic role of T3 in protecting general anesthetic induced neuronal injury in the developing brain is likely to occur through enhancing expression of PSD-95 and the NMDA NR2A and NR2B expression.

A narrative review of exploring potential salivary biomarkers in respiratory diseases: still on its way.

Saliva is abundant with proteins, metabolites, DNA, and a diverse range of bacterial species. During the past two decades, saliva has emerged as a novel diagnostic and evaluation medium for several diseases. Collection of saliva samples is simple, minimally invasive, and convenient even in infants, children, and patients with anxious. Furthermore, with the development of hypersensitive techniques [e.g., microsensor arrays, enzyme-labeled immunosensors, nanoparticle-labeled immunosensors, capacitive or impedimetric immunosensors, magneto immunosensors, field effect transistor immunosensors, and surface enhanced Raman spectroscopy (SERS)], the sensitivity and accuracy of saliva diagnostic procedures have been improved. Nowadays, saliva has been used as a potential medium for several disease diagnosis and assessment, such as periodontitis, caries, cancers, diabetes mellitus, and cardiovascular diseases. Saliva has been used widely for studying microbiomics, genomics, transcriptomics, proteomics, and metabolomics of respiratory diseases, however, the use of salivary biomarkers for the diagnosis, prognosis, and monitoring of respiratory disease is still in its infancy. Herein, we review the progress of research on salivary biomarkers related to several respiratory diseases, including bronchial asthma, chronic obstructive pulmonary disease (COPD), obstructive sleep apnea (OSA), pneumonia, tuberculosis (TB), Langerhans cell histiocytosis (LCH) and cystic fibrosis (CF). Furthermore, several limitations of saliva test such as the lack of standard protocol for saliva collection and reasonable reference values for saliva test are also mentioned in this review.

Prenatal sevoflurane exposure causes neuronal excitatory/inhibitory imbalance in the prefrontal cortex and neurofunctional abnormality in rats.

The balance of excitatory and inhibitory neurons in the central nervous system is critical for maintaining brain function and sevoflurane, a general anesthetic and an GABA receptor modulator, may change the balance of excitatory and inhibitory neurons in the cortex during early brain development. Herein, we investigated whether prenatal sevoflurane exposure (PSE) disturbs cortical neuronal development and brain function. Pregnant rats at the gestational day 14.5 were subjected to sevoflurane exposure at 3.0% for 3 h and their offspring were studied thereafter. We found a significant increase of parvalbumin-positive neurons, vesicular GABA transporter (VGAT) and GAD67 expression, and GABA neurotransmitter, and a significant decrease of vesicular glutamate transporter 1 (VGLUT1) expression and glutamate in the medial prefrontal cortex (mPFC) of offspring. Pyramidal neurons showed atrophy with shorter dendrites, less branches and lower spine density visualized by Golgi stain and a decrease of excitability with the increased miniature inhibitory postsynaptic current (mIPSC) frequency and amplitude, the decreased miniature excitatory postsynaptic current (mEPSC) frequency and excitation/inhibition (E/I) ratio using whole-cell recording in offspring. There was a significant increase of inhibitory synapse in the mPFC detected by electron microscopy. Furthermore, PSE animals showed hypo-excitatory phenotype including depression-like behaviors and learning deficits. Thus, our studies provide novel evidence that PSE causes the persisted imbalance of excitatory and inhibitory neurons in the mPFC, and this is very likely the mechanisms of the sevoflurane-induced brain functional abnormalities.

[Quercetin attenuates Ox-LDL-induced calcification in vascular smooth muscle cells by regulating ROS-TLR4 signaling pathway].

OBJECTIVE: To determine whether quercetin inhibits oxidized low-density lipoprotein (Ox-LDL)-induced osteogenic differentiation and calcification of vascular smooth muscle cells (VSMCs) and understand the underlying mechanism. METHODS: The calcification of human VSMCs following Ox-LDL treatment was assessed using alizarin red staining and by detecting ALP activity. The mRNA expressions of the bone-related genes including Msx2, BMP2 and Osterix, and the contractile proteins including SMA and SM22a were analyzed using qPCR. The effects of quercetin were investigated on OxLDL-induced VSMC calcification and changes in ALP activity, expressions of Msx2, BMP2, Osterix, SMA and SM22a, ROS levels and SOD activity. The effect of Toll like receptor 4 (TLR4) silencing mediated by siRNA transfection on cell calcification, ALP activity, gene expressions and ROS levels were investigated. RESULTS: Ox-LDL treatment promoted VSMC calcification and up-regulated TLR4 expression. Quercetin treatment significantly attenuated Ox-LDL-induced VSMC calcification, reduced ALP activity, down-regulated the expression levels of Msx2, BMP2 and Osterix, and up-regulated the expressions of vascular smooth muscle contractile proteins SMA and SM22a. In addition, Quercetin treatment markedly increased SOD activity, reduced ROS levels and TLR4 expression in VSMCs. Silencing TLR4 expression using TLR4 siRNA also significantly decreased calcification of the VSMCs. CONCLUSIONS: Quercetin inhibits Ox-LDL-induced VSMC calcification in VSMCs possibly by targeting the ROS/TLR4 signaling pathway.

Precise ratiometric loading of PTX and DOX based on redox-sensitive mixed micelles for cancer therapy.

PTX and DOX have different anticancer mechanisms. The combination of the two anticancer drugs could synergically enhance their anticancer effect, but simultaneously accompanied by severe side effects. In the present study, we constructed a mixed micelle system based on redox-sensitive mPEG-SS-PTX and mPEG-SS-DOX conjugate. The drug delivery system has a fixed and high drug loading content of 24.2% (PTX∼14.8% and DOX∼9.4%) with a precise ratio of PTX and DOX to realize the synchronized and controlled release. The mixed micelle has an average size of 93.3nm with a narrow distribution, suitable for passive targeting to tumor tissues by the EPR effect. In vitro release profile and in vitro anticancer results show the mixed micelles have obvious redox-sensitive release properties in reducing environment and have a significant cytotoxicity to A549 and B16 cells. Importantly, in vivo study shows the mixed micelles have no obvious side effect on mice compared to free PTX/DOX samples during the treatment. Therefore, the constructed redox-sensitive mixed micelle is a promising drug delivery system for cancer therapy.

Testosterone promotes tube formation of endothelial cells isolated from veins via activation of Smad1 protein.

Testosterone (T) deficiency is positively correlated with the increased incidence of cardiovascular disease. However, the effects of T on vascular endothelial cells remain obscure. Tube formation capacity is critical for vascular regeneration/repair and Smad1 plays an important role in these events. In this study, we investigated the effects of T on Smad1 activation and tube formation of cultured human umbilical endothelial cells (HUVECs). Our results showed that T rapidly increased endothelial Smad1 phosphorylation. This effect was mimicked by cell-impermeable T-BSA conjugates and was not altered by transcriptional inhibitor actinomycin D or translational inhibitor cycloheximide. T-induced Smad1 phosphorylation was blocked by ERK1/2 and c-Src inhibitors or their specific siRNAs, while it was reinforced by ERK1/2 or c-Src overexpression. Indeed, T rapidly activated ERK1/2 and c-Src signalings and c-Src was confirmed as the upstream of ERK1/2. Moreover, caveolae disruptor methyl-ß-cyclodextrin (ß-MCD) blocked Smad1 activation induced by T. The association of caveolin-1 with androgen receptor (AR) or c-Src was detected by immunoprecipitation and it was significantly increased by rapid T stimulation. Furthermore, fractional analysis showed that AR and c-Src were expressed in caveolae-enriched membrane fractions. T promoted tube formation of HUVECs, which was blocked by c-Src and ERK1/2 inhibitors or by the knockdown of Smad1. In conclusion, T increased tube formation of endothelial cells isolated from veins by stimulating Smad1 phosphorylation in a nongenomic manner, which was mediated by signals from AR/c-Src located in caveolae to ERK1/2 cascade. These findings may shed new light on the relevance of T to its vascular functions.

Prenatal ketamine exposure causes abnormal development of prefrontal cortex in rat.

Ketamine is commonly used for anesthesia and as a recreational drug. In pregnant users, a potential neurotoxicity in offspring has been noted. Our previous work demonstrated that ketamine exposure of pregnant rats induces affective disorders and cognitive impairments in offspring. As the prefrontal cortex (PFC) is critically involved in emotional and cognitive processes, here we studied whether maternal ketamine exposure influences the development of the PFC in offspring. Pregnant rats on gestational day 14 were treated with ketamine at a sedative dose for 2 hrs, and pups were studied at postnatal day 0 (P0) or P30. We found that maternal ketamine exposure resulted in cell apoptosis and neuronal loss in fetal brain. Upon ketamine exposure in utero, PFC neurons at P30 showed more dendritic branching, while cultured neurons from P0 PFC extended shorter neurites than controls. In addition, maternal ketamine exposure postponed the switch of NR2B/2A expression, and perturbed pre- and postsynaptic protein expression in the PFC. These data suggest that prenatal ketamine exposure impairs neuronal development of the PFC, which may be associated with abnormal behavior in offsprings.

Autophagy Promotes Peripheral Nerve Regeneration and Motor Recovery Following Sciatic Nerve Crush Injury in Rats.

Autophagy maintains cellular homeostasis by stimulating the lysosomal degradation of cytoplasmic structures, including damaged organelles and dysfunctional proteins. The role of autophagy in the renewal and regeneration of injured peripheral nerves remains poorly understood. The current study investigated the role of autophagy in peripheral nerve regeneration and motor function recovery following sciatic nerve crush injury in rats by stimulating or suppressing autophagy and detecting the presence of autophagosomes and LC3-II expression by electron microscopy and Western blotting, respectively. Neurobehavioral function was tested by CatWalk gait analysis 1, 2, 3, and 6 weeks after injury, and the expression of neurofilament (NF)-200 and myelin basic protein (MBP) at the injury site was examined by immunocytochemistry. Apoptosis at the lesion site was determined by the terminal deoxynucleotidyl transferase dUTP nick end labeling assay. Treatment of injured rats with the autophagy inducer rapamycin increased the number of autophagosomes and LC3-II expression while reducing the number of apoptotic cells at the lesion; this was associated with an upregulation of MBP and NF-200 expression and increased motor function recovery as compared to sham-operated rats and those that were subjected to crush injury but untreated. The opposite effects were observed in rats treated with the autophagy inhibitor 3-methyladenine. These data indicate that the modulation of autophagy in peripheral nerve injury could be an effective pharmacological approach to promote nerve regeneration and reestablish motor function.

[Effect of ulinastatin for perioperative cardiac protection in elderly patients undergoing major gastrointestinal surgery].

OBJECTIVE: To study protective effect of ulinastatin on perioperative cardiac function in elderly patients undergoing major gastrointestinal surgery. METHODS: Sixty elderly patients (32 male and 28 female patients) aged 60-82 years scheduled for major gastrointestinal surgery were randomized into ulinastatin group and control group. The patients in ulinastatin group received 2×10(5) U ulinastatin rapidly administered via a intravenous pump immediately before operation with subsequent continuous infusion at the rate of 1×10(5) U until the completion of surgery, and those in the control group received the same amount of saline instead. In both groups, the mean arterial pressure (MAP), heart rate (HR), left ventricular ejection fraction (LVEF), and cardiac output (CO) were monitored immediately before surgery (T0) and at 1 h (T1), 2 h (T2) and 3 h (T3) after the start of surgery. The total dopamine dose used was recorded at the end of surgery, and blood samples were collected at T0 and at 6 h (T4) and 12 h (T5) after the operation for determination of serum levels of cTn, CK-MB and BNP. RESULTS: In both groups, MAP, LVEF and CO were significantly decreased at T2 and T3 (P<0.05) and serum levels of cTn, CK-MB and BNP significantly increased at T4 and T5 compared to those at T0 (P<0.05). Compared with the control group, the patients in ulinastatin group showed significantly higher MAP, LVEF and CO at T2 and T3 and lower serum levels of cTn, CK-MB and BNP at T4 and T5. CONCLUSION: Ulinastatin offers effective perioerative cardiac protection in elderly patients undergoing major gastrointestinal surgery.

[Determination of the 50% effective concentration of dexmedetomidine as an adjuvant in combined spinal-epidural anesthesia with Narcotrend].

OBJECTIVE: To determine the 50% effective concentration (EC(50) ) of dexmedetomidine (DEX) as an adjuvant for achieving the minimal effect in combined spinal-epidural anesthesia (CSEA) with Narcotrend. METHODS: Thirty ASA class I-II patients undergoing CSEA were allocated to receive 0.5% bupivacaine injection (2 ml) in the subarachnoid space, followed by a target-controlled infusion of DEX for at least 60 min. The target concentration of DEX was adjusted according to the response of the previous patient using a double-blind, up-and-down sequential method. The initial DEX concentration of the first patient was 0.5 ng/ml. An Observers Assessment of Alertness/Sedation Scale (OAA/S) score of no more than 3 within 30 min defined an effective sedation. The Narcotrend index (NTI) related to the OAA/S score was recorded as well. The correlation between Narcotrend and OAA/S score was analyzed. RESULTS: The EC(50) of DEX as an adjuvant in CSEA was 0.35∓0.07 ng/ml. The NTI was positively correlated to the OAA/S score (r=0.967, P=0.000). The regression equation was OAA/S =-3.922+0.094NTI (F=401.710, P=0.000). CONCLUSION: We suggest a novel protocol using DEX as a adjunct in CSEA. The EC(50) % of DEX to result in ideal sedation (OAA/S≤3) was 0.35∓0.07 ng/ml. NTI may serve as an objective index for sedation assessment in CSEA.

[Effect of the degree of muscle relaxation on motor-evoked potential elicited by transcranial electrical stimulation in spine surgery].

OBJECTIVE: To study the effect of the degree of muscle relaxation on motor-evoked potential elicited by transcranial electrical stimulation in patients undergoing spine surgery. METHODS: Sixty ASA I or II patients undergoing spine surgery were randomly divided into 5 groups (n=12). After an initial intubation, continuous cisatracurium infusion was administered with continuous monitoring of T1. The infusion dose was adjusted according muscle relaxation monitoring, and different muscle relaxation degrees were maintained in the 5 groups. The band and latency of D1 in motor-evoked potential was observed with also subjective assessment of the muscle relaxation. RESULTS: Significant differences in the band and latency were noted in groups I and II compared with the reference values, but not in groups III, IV and V. Subjective assessment revealed significant differences between groups IV and V and groups I and III in terms of the number of cases with poor muscle relaxation. CONCLUSION: T1 value between 10% and 15% is sufficient for MEP monitoring and allows the maintenance of good muscle relaxation during spine surgery.

[Influence of epidural ropivacaine in combination with fentanyl for labor analgesia on the clinical outcome of labor].

OBJECTIVE: To investigate the effect of epidural ropivacaine in combination with fentanyl for labor analgesia on the clinical outcome of labor. METHODS: A retrospective study was conducted involving 281 healthy primiparas, including 106 undergoing spontaneous labor who received epidural 0.15% ropivacaine in combination with fentany (1microg/ml) and 175 without epidural analgesia. The active phase duration, durations of each labor stages, delivery modes, management of labor, postpartum hemorrhage, incidence of fetal distress and asphyxia neonatorum were recorded in the two groups. The visual analogue scale (VAS) was used to assess the pain of uterine contraction, and modified Bromage scoring system applied to evaluate the lower limb motor block. RESULTS: There were no significant differences in the duration of the first, third or the total labor stages between the two groups, but the second labor stage was prolonged in the labor analgesia group. The ratio of spontaneous labor, assisted vaginal delivery, and incidence of asphyxia neonatorum were higher, whereas the duration of the active stage was shortened in the analgesia group. CONCLUSION: Epidural ropivacaine in combination with fentanyl in labor can decrease the incidence of cesarean section, and the duration of the active stage can be shortened with application of ocytocin.

Effects of isoflurane on the actions of neuromuscular blockers on the muscle nicotine acetylcholine receptors.

In this study, we tested the hypothesis that volatile anesthetic enhancement of muscle relaxation is the result of combined drug effects on the nicotinic acetylcholine receptors. The poly A m RNA from muscle by isolation were microinjected into Xenopus oocytes for receptor expression. Concentration-effect curves for the inhibition of Ach-induced currents were established for vecuronium, rocuranium, and isoflurane. Subsequently, inhibitory effects of NDMRs were studied in the presence of the isoflurane at a concentration equivalent to half the concentration producing a 50% inhibition alone. All tested drugs produced rapid and readily reversible concentration-dependent inhibition. The 50% inhibitory concentration values were 889 micromol/L (95% CI: 711-1214 micromol). 33.4 micromol (95% CI: 27.1-41.7 nmol) and 9.2 nmol (95% CI: 7.9-12.3 nmol) for isoflurane. rocuranium and vecuronium, respectively. Coapplication of isoflurane significantly enhanced the inhibitory effects of rocuranium and vecuronium, and it was especially so at low concentration of NMDRs. Isoflurane increases the potency of NDMRs, possibly by enhancing antagonist affinity at the receptor site.