BACKGROUND:Ca2+expression in astrocytes has been found to be closely related to cognitive function,and the Ca2+signaling pathway regulated by inositol 1,4,5-trisphosphate receptors(IP3R2)and ryanodine receptor(RYR)2 receptors has become a hot spot in the study of cognitive disorder-related diseases. OBJECTIVE:To investigate the expression of Ca2+signals mediated by IP3R2 and RYR2 in hippocampal astrocytes in animal models of delayed encephalopathy after acute carbon monoxide poisoning,and to explore the possible pathogenesis of delayed encephalopathy after acute carbon monoxide poisoning. METHODS:C57BL mice with qualified cognitive function were selected by Morris water maze experiment and randomly divided into control group and experimental group.An animal model of delayed encephalopathy after acute carbon monoxide poisoning was established by static carbon monoxide inhalation in the experimental group,and the same amount of air was inhaled in the control group.Behavioral and neuronal changes,astrocyte specific marker glial fibrillary acidic protein,IP3R2,RYR2 receptor and Ca2+concentration in astrocytes of the two groups were detected using Morris water maze,hematoxylin-eosin staining,western blot,immunofluorescence double labeling and Ca2+fluorescence probe at 21 days after modeling. RESULTS AND CONCLUSION:In the Morris water maze,the escape latency of the experimental group was significantly longer than that of the control group(P<0.05).Hematoxylin-eosin staining results showed that in the experimental group,the number of hippocampal pyramidal cells decreased,the cell structure was disordered,and the nucleus was broken and dissolved.Immunofluorescence results showed that IP3R2 and RYR2 were co-expressed with glial fibrillary acidic protein in the hippocampus,and the expressions of IP3R2,RYR2 and glial fibrillary acidic protein were up-regulated in the hippocampus of the experimental group(P<0.05).Western blot analysis showed that the expressions of IP3R2,RYR2,and glial fibrillary acidic protein in the hippocampus of the experimental group were increased(P<0.05).Ca2+concentration in hippocampal astrocytes increased significantly in the experimental group(P<0.05).To conclude,astrocytes may affect Ca2+signals by mediating IP3R2 and RYR2 receptors,then impair the cognitive function of mice with carbon monoxide poisoning,and eventually lead to delayed encephalopathy after acute carbon monoxide poisoning.

Three 2,3-diketoquinoxaline alkaloids were isolated from Heterosmilax yunnanensis Gagnep. Their structures were determined through 1D and 2D NMR, HR-ESI-MS, UV, and IR as 1-[5′-(3″-hydroxy-3″-methyl) glutaryl] ribityl-2,3-diketo-1,2,3,4-tetrahydro-6,7-dimethylquinoxaline (1), 1-[2′-(3″-hydroxy-3″-methyl) glutaryl]ribityl-2,3-diketo-1,2,3,4-tetrahydro-6,7-dimethylquinoxaline (2), and 1-ribityl-2,3-diketo-1,2,3,4-tetrahydro-6,7-dimethylquinoxaline (3). Compounds 1 and 2 are novel compounds, and 3 was isolated from H. yunnanensis for the first time. The hepatoprotective activity of these three compounds was evaluated, with compound 3 showing promising hepatoprotective activity.

Objective:To explore the clinical efficacy and safety of moderate-dose glucocorticoid treatment in patients with advanced thymoma.Methods:A retrospective analysis was conducted on clinical data of 56 patients with thymoma who received glucocorticoid treatment at Beijing Tongren Hospital, Capital Medical University, from January 2018 to March 2023. Among them, there were 33 males and 23 females; aged from 28 to 72 years old, with a median age of(43.8±11.2)years old. There were 16 cases of type B1 thymoma, 23 cases of type B2, and 17 cases of type B3. There were 17 cases with myasthenia gravis and 1 case with pure red blood cell aplasia. All patients had received radiotherapy or chemotherapy. Patients received moderate-dose glucocorticoid treatment(50 mg/day), and tumor response was evaluated by CT scan after 2 weeks of treatment. The tumor treatment effect and adverse reactions were recorded.Results:Among the patients, 22 cases achieved partial remission, and 3 cases achieved complete remission, resulting in an overall objective response rate (ORR) of 44.6%. Among them, 23 patients had type B2 thymoma, with 17 achieving partial remission and 3 achieving complete remission, resulting in an ORR of 87.0%. Among the 16 patients with type B1 thymoma, 5 achieved partial remission. No efficacy was observed in 17 patients with type B3 thymoma. The follow-up period ranged from 2 to 26 months, and 2 patients experienced thymoma recurrence.Conclusion:Moderate-dose glucocorticoid treatment demonstrates effective and safe outcomes in advanced or invasive thymoma, particularly for the treatment of type B2 thymoma.

Background/Aims: The histological characteristics and natural history of precirrhotic primary biliary cholangitis (PBC) with portal hypertension (PH) are unclear. Our aim was to clarify the prevalence, risk factors, and histological characteristics of precirrhotic PBC patients with PH. Methods: This retrospective study compared the clinical features, histological characteristics, and response to ursodeoxycholic acid (UDCA) between the PH and non-PH groups of precirrhotic PBC patients. Results: Out of 165 precirrhotic PBC patients, 40 (24.2%) also had PH. According to histological stage 1, 2 and 3 disease, 5.3% (1/19), 17.3% (17/98), and 45.8% (22/48) of patients also had PH, respectively. Precirrhotic PBC with PH was significantly positively correlated with bile duct loss, degree of cytokeratin 7 positivity, and degree of fibrosis in the portal area, but significantly negatively correlated with lymphoid follicular aggregation. Compared to the non-PH group, patients in the PH group showed a higher prevalence of obliterative portal venopathy, incomplete septal fibrosis, portal tract abnormalities and non-zonal sinusoidal dilatation (p<0.05). In addition, patients with PH were more likely to present with symptoms of jaundice, ascites, epigastric discomfort, a poorer response to UDCA, and more decompensation events (p<0.05). High alkaline phosphatase levels, low white blood cell counts, high Mayo scores, and high FIB-4 index values were risk factors for precirrhotic PBC with PH. Conclusions Approximately 24.2% of precirrhotic PBC patients have PH, which is histologically related to the injury of bile ducts. High alkaline phosphatase levels, low white blood cell counts, high Mayo scores, and high FIB-4 index values are associated with increased risk of precirrhotic PBC with PH.

OBJECTIVES: To estimate postmortem interval (PMI) by analyzing the protein changes in skeletal muscle tissues with the protein chip technology combined with multivariate analysis methods. METHODS: Rats were sacrificed for cervical dislocation and placed at 16 ℃. Water-soluble proteins in skeletal muscles were extracted at 10 time points (0 d, 1 d, 2 d, 3 d, 4 d, 5 d, 6 d, 7 d, 8 d and 9 d) after death. Protein expression profile data with relative molecular mass of 14 000-230 000 were obtained. Principal component analysis (PCA) and orthogonal partial least squares (OPLS) were used for data analysis. Fisher discriminant model and back propagation (BP) neural network model were constructed to classify and preliminarily estimate the PMI. In addition, the protein expression profiles data of human skeletal muscles at different time points after death were collected, and the relationship between them and PMI was analyzed by heat map and cluster analysis. RESULTS: The protein peak of rat skeletal muscle changed with PMI. The result of PCA combined with OPLS discriminant analysis showed statistical significance in groups with different time points (P<0.05) except 6 d, 7 d and 8 d after death. By Fisher discriminant analysis, the accuracy of internal cross-validation was 71.4% and the accuracy of external validation was 66.7%. The BP neural network model classification and preliminary estimation results showed the accuracy of internal cross-validation was 98.2%, and the accuracy of external validation was 95.8%. There was a significant difference in protein expression between 4 d and 25 h after death by the cluster analysis of the human skeletal muscle samples. CONCLUSIONS The protein chip technology can quickly, accurately and repeatedly obtain water-soluble protein expression profiles in rats' and human skeletal muscles with the relative molecular mass of 14 000-230 000 at different time points postmortem. The establishment of multiple PMI estimation models based on multivariate analysis can provide a new idea and method for PMI estimation.
Animals ; Humans ; Rats ; Multivariate Analysis ; Postmortem Changes ; Protein Array Analysis ; Technology

Objective: To investigate the current status of myopia in children and adolescents in Qamdo, Tibet, and analyze related influencing factors, so as to provide a basis for the prevention and control of adolescents in plateau areas. Methods: A cross sectional study was conducted among 959 children and adolescents randomly selected from one district and two counties in Qamdo (from the fourth grade of elementary school to the second grade of high school) for visual acuity and refraction tests and filled out a vision related behavior questionnaire to analyze the incidence of myopia among adolescents in the region and its associated factors. Results: The myopia rate of adolescents in grades 4-11 was 54.43%, the rate of undercorrection of refractive errors was 85.25%, and the percentage of students wearing eyeglasses was 34.67%,fully vision correction rate was 42.54%. The myopia rate of students in grades 4-6 was 35.14%, 64.71% in grades 7-9, and 73.48% in grades 10-11. The myopia rate increased with grades( χ 2= 101.18 , P <0.01). The myopia rate (70.40%) of urban students (grades 4-9) was higher than that of county level(41.45%), and the myopia rate of students with myopia from either parent (68.24%) was higher than that of students without myopia (51.91%) , the myopia rate of girls (59.96%) was higher than that of boys (48.36%)( χ 2=53.19，13.46，12.98， P <0.01). Use electronic products for more than 2.5 hours per day, electronic devices usage after bedtime, the light low indoor brightness when studying on a sunny day, and only use one of the table lamps or roof lights when studying at night, preference for fried food, poor sleep quality, in the morning the students who still feel tired are at higher risk of myopia( χ 2=10.35, 10.91, 6.87, 4.25, 4.97, 5.71, 12.11, P < 0.05). Multivariate regression analysis showed that the occurrence of myopia was related to region, grade, gender, parental myopia, time spent on electronic products every day in the past 5 months, and sleep quality( P <0.05). Conclusion The high rate of myopia in children and adolescents in Qamdo may be related to the quality of sleep, the length of time electronic products are used, the eye environment, and the frequency of eating fried foods. Outdoor activities do not show significant differences.

The present study was aimed to explore the involvement of dopamine D1 receptor of the anterior cingulate cortex (ACC) in the regulation of chronic inflammatory pain-related emotion. On the first day, the rats were acclimated to the environment and the baseline indices were measured. On the second day, the rats were administered with the dopamine D1 receptor antagonist SCH-23390 or agonist SKF38393 in the ACC, and then they were subcutaneously injected with complete Freund's adjuvant (CFA, 0.08 mL) in the left hind paw to establish conditioned place avoidance (CPA) response after pairing with specific environment. On the third day, the CPA response and the firing frequency of ACC neurons were observed synchronously, and the open-field behavior, mechanical pain behavior and paw withdrawal latency (PWL) tests were also observed subsequently. In other experiments, rats were given subcutaneous injection of normal saline (NS) on the left hind paw after SCH-23390 or SKF-38393 was administered in the ACC, and then the same observations were performed. The results showed that: (1) Compared with the control group, the PWL and mechanical pain thresholds of rats injected with CFA on the left hind paw were significantly decreased (P < 0.05); (2) The residence time of rats injected with CFA in the "pain environment" and open field center was significantly shortened (P < 0.05); (3) Pre-injection of antagonist SCH-23390 in ACC (10 μg) alleviated the anxiety-like negative behavior response induced by CFA (P < 0.05) and reversed CFA-induced increases of discharge frequency of ACC neurons (P < 0.05); (4) Pre-injection of agonist SKF-38393 in the ACC (10 μg) induced CPA-like behavioral response in rats injected with NS in the left hind paw, and increased the firing frequency of ACC neurons (P < 0.05); (5) Immunofluorescence detection showed that dopamine D1 receptor and NMDA receptor were co-expressed in the same neuron. These results suggest that inhibition of dopamine D1 receptor in ACC can alleviate the negative emotional response induced by persistent pain.
2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine/adverse effects* ; Animals ; Anxiety ; Chronic Pain ; Gyrus Cinguli ; Hyperalgesia ; Rats ; Receptors, Dopamine D1/metabolism*

Objective: To compare the difference between the built-in and external reference electrode of microwire electrode array in the process of recording rat brain neuron firings, optimizing the production and embedding of the microwire electrode array, and providing a more affordable and excellent media tool for multi-channel electrophysiological real-time recording system. Methods: A 16 channel microwire electrode array was made by using nickel chromium alloy wires, circuit board, electrode pin and ground wires (silver wires). The reference electrode of the microwire electrode array was built-in (the reference electrode and electrode array were arranged in parallel) or external (the reference electrode and ground wire were welded at both ends of one side of the electrode), and the difference between the two electrodes was observed and compared in recording neuronal discharges in ACC brain area of rats. Experimental rats were divided into built-in group and external group, n=8-9. The test indicators included signal-to-noise ratio (n=8), discharge amplitude (n=380) and discharge frequency (n=54). Results: The microwire electrode array with both built-in and external reference electrodes successfully recorded the electrical signals of neurons in the ACC brain region of rats. Compared with the external group, the electrical signals of neurons in built-in group had the advantages of a higher signal-to-noise ratio (P＜0.05), a smaller amplitude of background signals and less noise interference, and a larger discharge amplitude(P＜0.05); there was no significant difference in spike discharge frequency recorded by these two types of electrodes (P＞0.05). Conclusion: When recording the electrical activity of neurons in the ACC brain region of rats, the microwire electrode array with built-in reference electrode recorded electrical signals with higher signal-to-noise ratio and larger discharge amplitude, providing a more reliable tool for multi-channel electrophysiology technology.
Action Potentials/physiology* ; Animals ; Brain ; Electrophysiological Phenomena ; Microelectrodes ; Neurons ; Rats

Objective: To establish an improved method of separating microglia from aged rats and to observe the biological characteristics of spinal microglia of aged rats. Methods: Young SD rats (2 months) were used as control group. Single cell suspension of rat microglia were prepared by trypsin, trypsin substitutes or mechanical net rubbing method. Then, by assessing the purity and survival rate of cells, and observing the morphological characteristics and analyzing the inflammatory functional characteristics, we optimized the isolation and purification method of microglia from aged rats (20 months old) , and observed the functional characteristics of spinal microglia in aged rats. Results: The survival rate of cells digested by pancreatic enzyme was low(young rats 83%, aged rats 60%). Although the survival rate of mechanical net rubbing method was higher than that of pancreatic enzyme digest methods (95%), the cell acquisition rate was lower(young rats(0.207±0.020)×10⁶, aged rats(0.243±0.023)×10⁶). Trypsin substitute dissociation combining density gradient centrifugation method was the best way to get abundant, active and higher survival microglia, and the purity reached more than 85%. We used this method to separate microglia from spinal cord of rats. Compared with the young rats, the spinal cord tissue of old rats was larger, the digestive fluid volume was higher, but the digestion time was shorter. Compared with the young rats, the aged rat spinal microglia had larger and rounder cell body, fewer and shorter protrusions, it tended to be activated morphologically, the level of proinflammatory cytokine IL-1β of microglia in aged rats was lower, and the level of antiinflammatory factor IL-10 was higher. Conclusion: The method of trypsin substitute dissociation combined with density gradient centrifugation was successfully established to isolate and purify microglia from spinal cord of rats, the spinal microglia of old rats showed anti-inflammatory phenotype.
Animals ; Cytokines ; Microglia ; Rats ; Rats, Sprague-Dawley ; Spinal Cord ; Trypsin

OBJECTIVES: To investigate the effects of injury time, postmortem interval (PMI) and postmortem storage temperature on mRNA expression of glycoprotein non-metastatic melanoma protein B (Gpnmb), and to establish a linear regression model between Gpnmb mRNA expression and injury time, to provide aimed at providing potential indexes for injury time estimation. METHODS: Test group SD rats were anesthetized and subjected to blunt contusion and randomly divided into 0 h, 4 h, 8 h, 12 h, 16 h, 20 h and 24 h groups after injury, with 18 rats in each group. After cervical dislocation, 6 rats in each group were collected and stored at 0 ℃, 16 ℃ and 26 ℃, respectively. The muscle tissue samples of quadriceps femoris injury were collected at 0 h, 12 h and 24 h postmortem at the same temperature. The grouping method and treatment method of the rats in the validation group were the same as above. The expression of Gpnmb mRNA in rat skeletal muscle was detected by RT-qPCR. The Pearson correlation coefficient was used to evaluate the correlation between Gpnmb mRNA expression and injury time, PMI, and postmortem storage temperature. SPSS 25.0 software was used to construct a linear regression model, and the validation group data was used for the back-substitution test. RESULTS: The expression of Gpnmb mRNA continued to increase with the prolongation of injury time, and the expression level was highly correlated with injury time (P<0.05), but had little correlation with PMI and postmortem storage temperature (P>0.05). The linear regression equation between injury time (y) and Gpnmb mRNA relative expression (x) was y=0.611 x+4.489. The back-substitution test proved that the prediction of the model was accurate. CONCLUSIONS The expression of Gpnmb mRNA is almost not affected by the PMI and postmortem storage temperature, but is mainly related to the time of injury. Therefore, a linear regression model can be established to infer the time of injury.
Animals ; Rats ; Glycoproteins ; Linear Models ; Melanoma ; Membrane Glycoproteins/genetics* ; Postmortem Changes ; Rats, Sprague-Dawley ; RNA, Messenger/metabolism* ; Time Factors