Dopamine transporter in the ventral tegmental area modulates recovery from propofol anesthesia in rats.

OBJECTIVE(S): To investigate the role of the dopamine transporter (DAT) in the ventral tegmental area (VTA) in the recovery from propofol anesthesia in rats. MATERIALS AND METHODS: A total of 150 Sprague-Dawley (SD) rats were randomly split into a normal control group (NC), saline group (S), propofol anesthesia group (P), adeno-associated viral-NC-mCherry (AAV-NC) group, and AAV-DAT-RNAi (DAT-RNAi) group (n = 30 per group). In rats in the AAV intervention group, AAV was injected into the VTA nucleus via a stereotaxer. The rats in each group were continuously pumped with propofol through the tail vein at a dose of 70 mg/kg/h, and the control group was infused with the same dose of saline at the same speed for 30 min. Immunofluorescence staining was used to observe the expression of c-fos protein in the prefrontal cortex (PFC). The induction and recovery time of propofol anesthesia were recorded based on the time of disappearance of the righting reflex (LORR) and recovery (RORR). The anesthesia depth score was performed on all rats 10 min after starting the administration and 10 min after withdrawal, which represented the depth of anesthesia during anesthesia and the degree of recovery during anesthesia recovery, respectively. electroencephalogram (EEG) was recorded during propofol anesthesia and recovery. RESULTS: Compared to the NC group, the RORR of the DAT-RNAi group was shortened, and the anesthesia depth score was higher (P < 0.05). In the DAT-RNAi group, during the period of propofol anesthesia, the ß wave frequencies increased, the θ wave frequencies decreased, and the expression of c-fos protein in PFC increased and during the recovery from propofol anesthesia, the α wave and ß wave frequencies were increased (P < 0.05). CONCLUSION: Knockdown of the DAT in the VTA region can enhance the activity of PFC neurons and promote the recovery of rats from propofol anesthesia.

Neuritin attenuates oxygen-glucose deprivation/reoxygenation (OGD/R)-induced neuronal injury by promoting autophagic flux.

The autophagy/apoptosis interaction has always been a focus of study in pathogenicity models. Neuritin is a neurotrophic factor that is highly expressed primarily in the central nervous system. Our previous study revealed that it protects against apoptosis in cortical neurons subjected to oxygen-glucose deprivation (OGD)/reoxygenation (OGD/R), and later animal experiments revealed that it can increase the expression of the autophagy-related protein LC3. Whether this neuroprotective effect is closely related to autophagy is still unclear. In this study, we hypothesized that neuritin can promote autophagic flux to protect nerve cells after OGD/R. To verify this hypothesis, we induced OGD/R in primary cortical neurons and assessed cell viability by the CCK8 and LDH assays. Cell apoptosis was assessed by Annexin V-FITC/PI, staining, and the contents and mRNA abundances of the autophagy-related proteins LC3 and p62, the apoptotic protein Caspase3 were quantified by Western blotting and RT-PCR. Autophagic flux was assessed by immunofluorescence after RFP-GFP-LC3 virus transfection, and ultrastructural changes in autophagosomes were observed by transmission electron microscopy (TEM). The results showed that cell viability was decreased, apoptosis was increased and autophagy was enhanced after OGD/R. Neuritin significantly increased cell viability, decreased apoptosis, further increased the expression of the autophagic flux-related protein LC3, further decreased p62 expression, and significantly increased the autophagosome number and autophagosome to lysosome ratio. Bafilomycin A1 (BafA1) is a late autophagy inhibitor, aggravated cell damage and apoptosis and counteracted the enhancement of autophagy activation and protective effects of neuritin. In conclusion, neuritin may promote the completion of autophagic flux by ameliorating neuronal damage after OGD/R.

Detection of autophagic flux in primary cerebral cortical neurons after oxygen glucose deprivation/reperfusion (OGD/R) using various methods.

The current research hot spot in the field of autophagic flux is to explain and alleviate disease from the perspective of autophagy. A highly sophisticated, sensitive, quantifiable and comprehensive method is required to accurately determine the dynamic process of autophagic flux. There are very few methods in neuroscience that specifically examine autophagic flux. Therefore, primary cortical neurons were divided into oxygen glucose deprivation/reperfusion (OGD/R) (group A) and OGD/R plus bafilomycin A1 (BafA1) (group B) groups. ① Transfection of the LC3 gene with the RFP-GFP tandem fluorescent label was performed. ② Direct quantification was performed using transmission electron microscopy (TEM). ③ Autophagy-related tools were used to detect the transformation of LC3I/II. ④ SQSTM1/P62 combined with the LC3 protein flip test was performed to comprehensively evaluate autophagic flux. Using method one, the ratio of autophagolysosomes to autophagosomes in group A was significantly increased based on fluorescence microscopy analysis. Using method two, the autophagy process in group A was more continuous and unobstructed based on TEM analysis, while only some partial processes were observed in group B, and the number of autophagosomes and autophagy lysosomes in group A was significantly greater more than that in group B. The LC3II/I ratio measured in method three was analysed in detail to explain the autophagic flux. The ratio of soluble p62 combined with the ratio of LC3II/I detected using method four reflected the activation of autophagy. In summary, each method has its own advantages, and different methods and indicators can be used to monitor different stages of autophagy. An understanding of these advantages and mastery of these methods, is a very promising strategy to systematically and objectively study central nervous system diseases, facilitate the rational use of drugs, and formulate effective treatment plans from the perspective of autophagy.

Dexmedetomidine and Netrin-1 Combination Therapy Inhibits Endoplasmic Reticulum Stress by Regulating the ERK5/MEF2A Pathway to Attenuate Cerebral Ischemia Injury.

The complexity of hard-to-treat diseases such as ischemic stroke strongly undermines the therapeutic potential of available treatment options. Therefore, current developments have gently shifted from a focus on monotherapy to combined or multiple therapies. Both dexmedetomidine and Netrin-1 have anti-neuronal apoptosis effects, but the mechanism is still unclear. The study aimed to estimate the efficacy of dexmedetomidine and Netrin-1 combination therapy against ERS-induced apoptosis after cerebral ischemia injury in vivo and in vitro, and whether the mechanism is related to the ERK5/MEF2A pathway. Adult male Sprague-Dawley rats were subjected to middle cerebral artery occlusion (MCAO) in vivo, 90 min ischemia and 24 h reperfusion. The hippocampus slices used to establish oxygen-glucose deprivation (OGD) injury model in vitro. Neterin-1 and Dexmedetomidine were pretreated and post-treated, respectively, before and after the model establishment. MEF2A knockdown was performed by microinjection of AAV9-MEF2A RNAi vector. Orthodromic population spike (OPS) at the end of reoxygenation were recorded. Neurobehavioral tests, TTC staining, Nissl staining, TUNEL staining were performed to assess the effect of the drugs. The expression of CHOP, GRP78, MEF2A, ERK5, and p-ERK5 were investigated by Western blot and immunofluorescence staining. Neurological deficit score, infarct volume, the expression of GRP78, CHOP, and neural apoptotic rate of MCAO group increased markedly. Combination of dexmedetomidine and Netrin-1 resulted in lower infarct volumes and fewer neurological impairments, higher OPS recovery rate, and less damaged and apoptotic cells after cerebral ischemia injury. Furthermore, expression levels of GRP78 and CHOP decreased in the combination therapy group, and it was more effective than the single drug group. Meanwhile, Combination of dexmedetomidine and Netrin-1 increased MEF2A expression and promoted ERK5 phosphorylation. However, the protective effect of dexmedetomidine combined with Netrin-1 in improving neurological function was significantly eliminated by pre-knockdown MEF2A. The neuroprotective effects of dexmedetomidine combined with Netrin on cerebral ischemia-reperfusion injury and hippocampal hypoxia injury in terms of ERS. The synergistic effect of combination therapy is related to the activation of ERK5/MEF2A signaling pathway.

Circular RNAs are abundant and dynamically expressed during the embryonic lung development of C57BL/6 mice.

Circular RNAs (circRNAs), a novel type of endogenous RNAs, can function as microRNA (miRNA) sponges capable of regulating gene transcription, binding to RNA-associated proteins, and even encoding proteins. CircRNAs are involved in various cell behaviors, such as proliferation and apoptosis. The mouse model has also been demonstrated to be similar to that of humans in many studies. To explore the profile of circRNAs during embryonic lung development and their potential functions in lung development-related diseases, mouse embryos at the pseudoglandular phase, canalicular phase, saccular phase, and alveolar phase were collected. High-throughput sequencing was then used to identify a total of 1,735 circRNAs (junction reads ≥5 and p < 0.05). It is well known that the functions of circRNAs are related to host genes. In our study, bioinformatics analysis indicated that the screened host genes were closely associated with lung development and included the Hippo signaling pathway, PI3K-Akt signaling pathways, and TGF-ß signaling pathways. Moreover, miRNA sponges are another mechanism involved in lung development. Therefore, we predicted many miRNAs binding to circRNAs, such as miR-17 and miR-20, using the TargetScan and miRanda databases. Previously, miRNAs were proven to be necessary for lung development. The peak expression of circRNAs is distributed at different time points, suggesting their involvement in different stages of embryonic mouse lung development.

Correlation between Preoperative Anxiety and ABO Blood Types: Evidence from a Clinical Cross-Sectional Study.

Gene-environment interaction is identified as the determinant in anxiety. ABO blood types represent a part of the genetic phenotype. Therefore, we assume ABO blood types correlate with preoperative anxiety. This cross-sectional study enrolled 352 patients with different ABO blood types, scheduled for elective surgery between 2018 and 2019 in the First Affiliated Hospital of Shihezi University. HADS (hospital anxiety and depression scale) scores and VA (visual analogue scales for anxiety) scores were all used to assess the preoperative anxiety in the A, B, AB, and O groups. Bivariate correlation and logistic regression were performed to identify relationships between preoperative anxiety and related variables. A significant difference in VA and HADS-A (anxiety) scores was found between the AB and other groups. The ratio of preoperative anxiety was 3.73 (95% CI [confidence interval]: 2.32-6.00, P < 0.001) times in female than in male; 0.36 (95% CI: 0.21-0.63, P < 0.001) times in ASA (American Society of Anesthesiologists) grade II than in grade I; 0.41 (95% CI: 0.20-0.86, P < 0.05) times in ASA grade III than in grade I; 1.25 (95% CI: 1.1-1.41, P < 0.001) times in higher VAS (visual analogue scales for pain) scores than in lower VAS scores; and 0.28 (95% CI: 0.16-0.49, P < 0.01) times in non-AB blood type than in AB blood type. Differences in ABO blood types were found in preoperative anxiety, and the AB group displayed a high preoperative anxiety level. ABO blood types, sex, ASA grade, and VAS were associated with preoperative anxiety. This trial is registered with ChiCTR1800019390.

The association between the expression of PAR2 and TMEM16A and neuropathic pain.

Chronic constriction injury (CCI) of the sciatic nerve may induce dorsal root ganglion (DRG) neuronal hyperexcitability and behaviorally expressed hyperalgesia. CCI is a model of neuropathic pain. To investigate the association between the expression of protease activated receptor 2 (PAR2), TMEM16A and neuropathic pain, the expression of PAR2 and TMEM16A proteins in the DRG neurons of rats following CCI of the sciatic nerve was investigated. Following the creation of the CCI model, the thermal withdrawal latency (TWL) was examined by a hot plate test. An immunofluorescence assay and western blot assay were performed to determine the expression of PAR2 and TMEM16A proteins in the ipsilateral L4­6 DRG neurons. The concentration of inositol 1,4,5­triphosphate (IP3) in the L4­6 DRG was determined by ELISA. In the CCI­D7 (7 days after CCI) and CCI­D14 (14 days after CCI) treatment groups, the TWL of rats was significantly shorter than that in the sham operated group (P<0.01; n=12). The expression of PAR2 and TMEM16A proteins in the CCI­D7 and CCI­D14 groups were significantly upregulated compared with the sham operated group (P<0.05; n=12). Additionally, it was revealed that PAR2 and TMEM16A were co­expressed in DRG neurons. It was also observed that IP3 significantly increased in the CCI­D7 and CCI­D14 groups compared with the sham operation group (P<0.05; n=6) as PAR2 and TMEM16A also increased. These findings suggest that the upregulation of PAR2 and TMEM16A in DRG neurons, the co­expression of the two proteins and increasing IP3 are critical to the development of neuropathic pain.