BACKGROUND:Primary cortical neurons and microglial cells play a crucial role in exploring cell therapies for neurological disorders,and most of the current methods for obtaining the two types of cells are cumbersome and require separate extraction.It is therefore crucial to find a convenient and rapid method to extract both types of cells simultaneously. OBJECTIVE:To explore a novel method for simultaneous extraction of primary cortical neurons and microglial cells. METHODS:Newborn suckling SD rats were taken within 24 hours.The brain was removed and placed in a dish with DMEM,and the pia mater was removed for later use.Primary neurons were extracted from the same brain tissue,and then the remaining brain tissue was used to extract microglial cells.The whole process was performed on ice.Extraction and culture steps of primary cortical neurons:The cerebral cortex was taken 2.0-3.0 mm with forceps,and the tissue was digested with papain for 20 minutes.After aborting digestion,the blown tissue presented an adherent tissue suspension.The supernatant cell suspension was obtained,filtered,and dispensed into 15 mL centrifuge tubes.After centrifugation and re-suspension,the cells were inoculated onto 6-well plate crawls coated with L-polylysine.Neuronal morphology was observed at 1-day intervals,and staining could be performed for identification using immunofluorescence staining of MAP2 and β-Tubulin by day 7.Microglia extraction and culture steps:The remaining brain tissue at 8-10 mm thick was subjected to microglial cell extraction,digested by trypsin for 20 minutes.After digestion was stopped,the tissue was blown to a homogenate,and then the homogenate was transferred to the culture bottle for culture.On day 14,the culture flasks were sealed and subjected to constant temperature horizontal shaking for 2 hours.Microglial cells were shed in the supernatant.Purified microglial cells were taken and continued to be cultured for 3 days for identification by Iba1 immunofluorescence staining. RESULTS AND CONCLUSION:(1)After 24 hours of culture,the neurons were adherent to the wall,the cytosol was enlarged,and some neurons developed synapses.After 3 and 5 days of culture,the cytosol was further enlarged,and most of the neurons were in the form of synapses,and some neurons were growing in clusters.On day 7,neuronal synapses were prolonged and thickened,and they were connected with each other to form a network.The neurons were identified by β-Tubulin and MAP2 immunofluorescence staining.(2)The cells grew close to the wall on day 1 of culture.On days 3,5,and 7,the density of microglial cells was small,and the cell morphology was bright oval or round,but the cells basically grew in clumps on the upper layer of other cells.On day 10,the density of microglial cells increased significantly.On day 14,microglial cells grew in dense clumps on the upper layer of other cells,and then they could be isolated and purified.The isolated and purified cells were taken and re-cultured to day 3 and identified as microglial cells by Iba1 immunofluorescence;their purity was greater than 95%.(3)The results show that primary cortical neurons and microglial cells obtained by this method after extraction and culture are of high purity,good morphology,and high viability.

BACKGROUND:Studies have found that activation of nuclear factor-erythroid 2-related factor 2/heme oxidase-1(Nrf2/HO-1)pathway can alleviate oxidative stress caused by cerebral ischemia-reperfusion injury,but whether human bone marrow mesenchymal stem cells(hBMSC)can activate Nrf2/HO-1 pathway to alleviate cerebral ischemia-reperfusion injury is still lacking relevant studies. OBJECTIVE:To investigate whether intracranial transplantation of hBMSC alleviates oxidative stress injury in cerebral ischemia-reperfusion animal models by activating Nrf2/HO-1 pathway. METHODS:Totally 40 male SPF SD rats were randomly divided into sham operation group,model group,hBMSC transplantation group,hBMSC+solvent group and hBMSC+Nrf2 inhibitor group.Each group consisted of eight animals.In the model group and the hBMSC transplantation group,middle cerebral artery occlusion model was prepared by thread embolization method.The thread embolization was removed 1 hour later,and 30 μL PBS or hBMSC cultured to at least passage 5 was injected into the right cortex and striatum of rats.In the hBMSC+Nrf2 inhibitor group and hBMSC+solvent group,the left ventricle was injected with Nrf2 inhibitor Brusatol and its solvent dimethyl sulfoxide respectively 24 hours before model establishment,then the middle cerebral artery occlusion model was prepared,and hBMSC was injected.Relevant indexes were detected 3 days after transplantation. RESULTS AND CONCLUSION:(1)CT and TTC staining showed the same area and volume of cerebral infarction:model group>hBMSC+Nrf2 inhibitor group>hBMSC+solvent group>hBMSC transplantation group>sham operation group.(2)Hematoxylin-eosin staining and Nissl's staining showed that the ischemic brain tissue was intact and the neurons were normal in the sham operation group.Compared with the model group,the pathological morphology and neuronal injury of the hBMSC transplantation group and the hBMSC+solvent group were significantly improved.Compared with the hBMSC+solvent group,the hBMSC+Nrf2 inhibitor group had more serious pathological morphology and neuronal damage.(3)Western blot assay and oxidative stress index detection results showed that compared with the sham operation group,Nrf2 and HO-1 proteins were decreased(all P<0.05),malondialdehyde was increased and superoxide dismutase was decreased(all P<0.05)in the model group.Compared with the model group,the expression levels of Nrf2 and HO-1 proteins were increased(all P<0.05),malondialdehyde was decreased and superoxide dismutase was increased(all P<0.05)in the hBMSC transplantation group and the hBMSC+solvent group.Compared with the hBMSC+solvent group,the expression levels of Nrf2 and HO-1 proteins were simultaneously decreased(all P<0.05),and malondialdehyde was increased and superoxide dismutase was decreased(all P<0.05)in the hBMSC+Nrf2 inhibitor group.(4)These results indicate that hBMSC can alleviate cerebral ischemia-reperfusion injury possibly by activating Nrf2/HO-1 pathway.

Objective: To examine the mediating effects of mindfulness level on resilience and symptoms of anxiety and depression among healthcare workers, so as to provide the reference for developing effective psychological intervention. Methods: The clinical doctors, nurses and public health professionals were selected using the stratified random cluster sampling method from hospitals, disease prevention and control centers, and health departments in five cities in Shandong Province, including Qingdao, Jinan, Rizhao, Jining and Liaocheng in January 2023. Basic information, mindfulness level, resilience and symptoms of anxiety and depression among healthcare workers were collected using general demographic questionnaires, the 5-item Mindful Attention Awareness Scale, 10-item Connor-Davidson Resilience Scale, Generalized Anxiety Disorder Scale and Patient Health Questionnaire, respectively. The Process program was used to analyze the mediating effects of mindfulness level on resilience and symptoms of anxiety and depression. Results: A total of 1 836 healthcare workers were investigated, including 472 males (25.71%) and 1 364 females (74.29%), and the median age was 39 (interquartile range, 12) years. There were 629 clinical doctors (34.26%), 963 nurses (52.45%) and 244 public health professionals (13.29%). The median scores of mindfulness level and resilience were 22 (interquartile range, 7) and 20 (interquartile range, 4) points, respectively. The detection rates of anxiety and depression symptoms were 49.78% and 72.28%, respectively. The mediation analysis showed that mindfulness level exerted a partial mediating effect between resilience and anxiety symptoms (β=-0.510, P<0.001), with a direct effect value of -0.130 and a mediating effect value of -0.046, and the mediating effect accounted for 26.14% of the total effect; mindfulness level also exerted a partial mediating effect between resilience and depression symptoms (β=-0.575, P<0.001), with a direct effect value of -0.120 and a mediating effect value of -0.052, and the mediating effect accounted for 30.23% of the total effect. Conclusion Mindfulness level plays a mediating effect between resilience and symptoms of anxiety and depression among healthcare workers.

Cell mechanics is essential to cell development and function,and its dynamics evolution reflects the physiological state of cells.Here,we investigate the dynamical mechanical properties of single cells under various drug conditions,and present two mathematical approaches to quantitatively character-izing the cell physiological state.It is demonstrated that the cellular mechanical properties upon the drug action increase over time and tend to saturate,and can be mathematically characterized by a linear time-invariant dynamical model.It is shown that the transition matrices of dynamical cell systems signifi-cantly improve the classification accuracies of the cells under different drug actions.Furthermore,it is revealed that there exists a positive linear correlation between the cytoskeleton density and the cellular mechanical properties,and the physiological state of a cell in terms of its cytoskeleton density can be predicted from its mechanical properties by a linear regression model.This study builds a relationship between the cellular mechanical properties and the cellular physiological state,adding information for evaluating drug efficacy.

Background Lipid metabolism in liver shows circadian-dependent profiles. The hepatotoxicity of environmental chemicals is dependent on circadian time. Objective To observe the effects of bisphenol A (BPA) exposure at different zeitgeber time (ZT) on hepatic and blood lipid metabolism and decipher the underlying mechanisms related to circadian rhythm in mice. Methods Thirty-five female C57BL/6J mice were sacrificed every 4 h in a light-dark cycle (12 h/12 h). The liver tissues were collected to describe the circadian profiles of hepatic Rev-erba, Bmal1, Clock, Srebp1c, and Chrebp mRNA expression levels within 24 h. Thirty female mice were divided into 6 groups by the timing (ZT3 represents the 3 h after light on, ZT15 represents the 3 h after light off) and dose (50 or 500 μg·kg⁻¹·d⁻¹) of BPA exposure to observe hepatotoxicity. Mice were gavaged with designed doses of BPA once per day for 4 weeks. Mice were maintained with ad libitum access to food and water and measured body weight weekly. After the experiment, mice were euthanatized and liver tissues were separated to determine the biochemical indicators of lipid metabolism and lipid metabolism- and circadian-related gene mRNA expressions. Results Hepatic Rev-erba, Bmal1, Clock, Srebp1c, and Chrebp mRNA expression levels were rhythmic during a 24 h period in mice. At ZT3 and ZT15, BPA did not alter body weight, plasma glucose, plasma total cholesterol, plasma low density lipoprotein cholesterol, and plasma triglycerides (P>0.05). The plasma high density lipoprotein cholesterol decreased in the 50 μg·kg⁻¹·d⁻¹ BPA group at ZT3 by 14.56% compared with the control group (P<0.05). The liver triglycerides increased in the 50 μg·kg⁻¹·d⁻¹ BPA group at ZT15 by 115.20% compared with the control group (P<0.05). BPA decreased Srebp1c mRNA expression level when dosing at ZT3 and increased Chrebp, Srebp1c, and Acc1 mRNA expression levels when dosing at ZT15 compared with the control group (P<0.05). BPA increased Bmal1 mRNA expression level and decreased Rev-erbα mRNA expression level at ZT3 exposure and decreased Bmal1 and increased Rev-erbα mRNA expression level at ZT15 exposure (P<0.05). Conclusion BPA exposure at light or dark period has different effects on hepatic lipid metabolism in mice. Hepatic lipid deposit appears when BPA is dosed at dark period. Rev-erbα-Bmal1 regulation circuits and the subsequent upregulation of Srebp1c and Chrebp and the target gene Acc1 may be involved.

Objective:To evaluate the clinical efficacy of the bridge pedicled transplantation of medial leg fascial flap combined with medial hemisoleus muscle flap for contralateral leg soft tissue defect.Methods:Between January of 2012 and January of 2018, 12 patients with soft tissue defect of the leg were treated with bridge pedicled transplantation of contralateral medial leg fascial flap combined with medial hemisoleus muscle flap by posterior tibial artery. There were 9 males and 3 female, aged from 19 to 53 years (mean, 35 years). The size of the soft-tissue defects ranged from 12 cm×8 cm to 18 cm×9 cm. The immediate coverage of the fascial and muscle flaps and vessel pedicle were repaired by a meshed split-thickness skin graft. The donor site was closed directly. After the transplantation of the one pedicle and two flaps survived, vascular pedicle was cut off.Results:All the fascial and muscle flaps survived completely. No clinical vascular deficiency was found on the fascial and muscle flaps postoperatively. One case developed distal muscle flap small skin graft necrosis, and spontaneous healed after 2 weeks of dressing change. Follow-up period ranged from 2.5 to 4.5 years (mean 3.8 years). A good contour was confirmed both at the recipient and donor sites. Satisfactory clinical results were obtained in this series.Conclusion:This method is suitable for the treatment of soft tissue defects of the leg with only one major blood vessel, which reduces the damage to the donor site.

Objective:To evaluate the efficacy of the repairing anterior tibial adjacent double wounds by transposition with single-pedicle two flaps of the medial head of gastrocnemius.Methods:Between January of 2012 and January of 2018, 10 patients with the anterior tibial adjacent double wounds (7 males and 3 female, aged from 21 to 45 years) were treated by transposition with single-pedicle two flaps of the medial head of gastrocnemius. The size of the soft-tissue defects ranged from 2.0 cm×2.5 cm to 4.5 cm×4.0 cm. The medial head of the gastrocnemius was divided into two flaps with a single pedicle to repair two adjacent wounds of the anterior tibial. The muscle flaps were immediately covered by a meshed split-thickness skin graft, and the wound in donor site was closed directly.Results:All the muscle flaps survived completely. No clinical vascular deficiency was found on the muscle flaps postoperatively. Small wound dehiscence was developed in one patient and spontaneously healed 2 weeks after dressing change. Patients were followed up for 2.0 to 4.5 years. A good contour was confirmed at the recipient and donor sites. Satisfactory clinical results were obtained in this series.Conclusion:This method is suitable for the repair of two adjacent small wounds of the anterior tibial which can reduce the damage to the donor site.

Objective: To explore the analgesic effect of electroacupuncture on neuropathic pain and the role of PI3K/AKT pathway in microglial activation in spinal cord. Methods: Chronic constriction injury model was established using Sprague-Dawley rats to mimic neuropathic pain. The pain intensity was then detected by paw withdrawal threshold and thermal withdrawal latency. Real-time quantitative PCR (qRT-PCR), Western blot were used to evaluate the activation of PI3K/AKT pathway as well as microglia in L4-5 of all rats. Proinflammatory cytokines TNF-α and IL-6 were analyzed by Enzyme-linked immunosorbent assay (ELISA). Results: It was demonstrated that chronic constriction injury-induced hyperalgesia was significantly improved by LY294002 intrathecal administration and electroacupuncture stimulation at “Zusanli” 足三里 (ST36) and “Yanglingquan” 阳陵泉 (GB34). Also, treatment with electroacupuncture significantly reduced the activation of microglia and downregulated the levels of TNF-α and IL-6, which was similar to the outcomes of LY294002 intrathecal administration. Furthermore, the expression and phosphorylation of PI3K/AKT signaling was markedly suppressed by electroacupuncture treatment. Conclusions: These findings indicated that electroacupuncture exhibited the analgesic effect on CCI rats by inhibiting the activation of microglia and production of proinflammatory cytokines in spinal cord through blocking the microglial PI3K/AKT signaling activation

Four hundred and twenty two patients with ureteral calculi undergoing surgical treatment from January 2016 to December 2016 were enrolled in the study.Urine samples were taken from upper and lower ureter of stone obstruction,pathogen examination and drug susceptibility tests were performed.Twenty nine strains of pathogens were isolated from the upper segment of ureter with a detection rate of 6.9%;22 strains were gram-negative bacteria(75.9%)and 3 strains were gram-positive bacteria(10.3%)and 4 strains were fungi(13.8%).Forty eight strains of pathogenic bacteria were isolated from the lower segment with a detection rate of 11.4%;37 strains were gram-negative bacteria(77.1%),11 strains were gram-positive bacteria(22.9%),no fungi was isolated.In 20 cases the positive results were obtained only from upper ureter urine samples, and in 39 cases the positive results were obtained only from lower segment samples.The same pathogens were detected from both upper and lower ureter of stone obstruction in 7 cases, and different pathogens were identified in 2 cases.The most common pathogens were Escherichia coli, followed by Proteus mirabilis, Klebsiella pneumoniae, Enterococcus faecalis and Enterococcus faecium.The resistance to quinolones in gram-negative bacteria was higher than that to cephalosporins.The resistance rate of Escherichia coli to cephalosporin was 36.7%-63.3%,that to ciprofloxacin and levofloxacin was 86.7%-100.0%; the resistance rate of Enterococcus to erythromycin was 100.0%.It is suggested that ureteral calculi obstruction may lead to negative culture results of conventional mid-stream urine samples.It is of clinical value to investigate the distribution and antibiotic resistance of pathogens both from upper and lower segments of ureter in patients with ureteral calculi.

Objective:To study the pharmacological effects of Leshibao chewable tablets for juvenile rats with anorexia. Methods:The rats were divided into 6 groups with 10 ones in each and fed with special forage for 4 weeks to establish the anorexia model of ju-venile rats. After the model establishment, from the 8th day, the treatment groups received Leshibao chewable tablets respectively at the dose of 0. 60, 0. 30 and 0. 15 g·kg-1 ·d-1 for three weeks. The general situation was observed, and the appetite and body weight were recorded during the treatment. At the end of experiment, the gastric juice amount, free gastric acidity, total gastric acidity and pepsin activity were determined, TP, ALB, TCHO, MTL and GAS were determined by kit methods. Additionally, the mice were given Leshibao chewable tablets at different doses (0. 80, 0. 40, 0. 20 g·kg-1 ·d-1 ) for 7 days, the effects of Leshibao chewable tablets on gastric emptying and intestinal propulsive function were observed. Results: Leshibao chewable tablets could significantly increase the body weight, appetite (P<0. 05 or P<0. 01), gastric juice amount, free gastric acidity , total gastric acidity and pepsin secretion (P<0. 05 or P<0. 01), significantly reduce TP, ALB and TCHO in serum (P<0. 05 or P<0. 01), significantly increase MTL and GAS contents (P<0. 05 or P<0. 01), and significantly promote gastric emptying and intestinal propulsion in mice (P<0. 05 or P<0. 01). Conclusion:Leshibao chewable tablets can significantly increase body weight and appetite, and improve digestive function re-markably, which exhibit excellent therapeutic effect in the anorexia model of juvenile rats.