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.

Idiopathic thrombocytopenic purpura (ITP) is a common bloody disease with a high incidence in children, but its diagnostic method is exclusive diagnosis, and the existing detection techniques are mostly invasive, which may cause secondary injury to patients and also may increase the risk of disease. In order to make up for the lack of the detection method, this study made a preliminary exploration on the diagnosis of children's ITP from the perspective of infrared thermography. In this study, a total of 11 healthy children and 22 ITP children's frontal infrared thermal images were collected, and the pattern characteristic (PFD), average temperature (Troi) and maximum temperature (MAX) characteristics of 7 target areas were extracted. The weighted PFD parameters were correlated with the platelet count commonly used in clinical diagnosis, and the sensitivity and specificity of the weighted PFD parameters for children's ITP were calculated through the receiver operating characteristic curve (ROC). The final results showed that the difference of the weighted PFD parameters between healthy children and ITP children was statistically significant, and the parameters negatively correlated with platelet count. Under the ROC curve, the area under the curve (AUC) of this parameter is as high as 92.1%. Based on the research results of this paper, infrared thermography can clearly show the difference between ITP children and healthy children. It is hoped that the methods proposed in this paper can non-invasively and objectively describe the characteristics of ITP infrared thermal imaging of children, and provide a new ideas for ITP diagnosis.
Area Under Curve ; Child ; Humans ; Platelet Count ; Purpura, Thrombocytopenic, Idiopathic ; Thermography

Objective To evaluate the effect of polydatin on neuropathic pain in rats.Methods Forty male Sprague-Dawley rats,aged 6-8 weeks,weighing 200-230 g,were randomly divided into 5 groups (n =8 each) using a random number table:sham operation group (group S),neuropathic pain group (group NP),polydatin 5 mg/kg group (group P1),polydatin 10 mg/kg group (group P2),and polydatin 20 mg/kg group (group P3).Neuropathic pain was induced by chronic constriction injury in NP and P1-3 groups.In group S,the sciatic nerve was only exposed but not ligated.In S and NP groups,normal saline 0.1 ml was injected intraperitoneally immediately after operation and at 1,3,5 and 7 days after operation (T1-4).In P1-3 groups,polydatin 5,10 and 20 mg/kg (in normal saline 0.1 ml) were injected intraperitoneally immediately after operation and at T1-4.At 1 day before operation (T0) and T1-4,the mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were measured.After measurement of pain threshold at T4,the rats were sacrificed,and L4-6 segments of the spinal cords were removed for determination of the expression of high-mobility group box 1 (HMGB1),Toll-like receptor 4 (TLR4),interleukin-1beta (IL-1β),tumor necrosis factor-alpha (TNF-α) and monocyte chemotactic protein-1 (MCP-1) by Western blot.Results Compared with group S,the MWT was significantly decreased,and the TWL was significantly shortened at T1-4 in group NP,the MWT was significantly decreased at T1-4,and the TWL was significantly shortened at T2-4 in group P1,the MWT was significantly decreased at T1-4,and the TWL was significantly shortened at T3.4 in group P2,the MWT was significantly decreased at T1-4 in group P3,and the expression of HMGB1,TLR4,IL-1β,TNF-α and MCP-1 was significantly up-regulated in NP,P1 and P2 groups (P＜0.05).Compared with group NP,the MWT was significantly increased at Tt-4,and the TWL was significantly prolonged at T1,2 in group P2,the MWT was significantly increased,and the TWL was significantly prolonged at T1-4 in group P3,the expression of HMGB1,TLR4,IL-1β,TNF-α and MCP-1 was significantly down-regulated in P2 and P3 groups (P＜0.05),and no significant change was found in the parameters mentioned above in group P1 (P＞0.05).Compared with group P1,the MWT was significantly increased at T4 in group P2,and the MWT was significantly increased at T14,the TWL was significantly prolonged at T3,4,and the expression of HMGB1,TLR4,IL-1β,TNF-α and MCP-1 was significantly down-regulated in group P3 (P＜0.05).Compared with group P2,the MWT was significantly increased at T3,4,and the expression of TLR4,IL-1β,TNF-α and MCP-1 was significantly down-regulated in group P3 (P＜0.05).Conclusion Polydatin can alleviate neuropathic pain through inhibiting inflammatory responses in the spinal cord of rats.

Objective To analysis the human cytomegalovirus (HCMV)and human T lymphotropic virus(HTLV)infection status in Beijing among voluntary blood donors.Methods Randomly selected 2 010 blood samples from five districts and counties of Beijing City to screen HCMV-IgG,HCMV-IgM and HTLV-Ⅰ/Ⅱ antibody by ELISA method.The positive samples were reexamined two times,two test results of samples were positive that were determined positive by ELISA. HTLV positive samples was confirmed by nested PCR.Results The HCMV-IgM and HCMV-IgG positive rates of Beijing blood donors were 2.19% and 92.59%,screened 1 case of anti-HTLV positive by ELISA method,then confirmed to be neg-ative result by nested PCR.The statistics showed that the HCMV-IgG positive rate female blood donors was higher than male (P <0.05).The positive rates of HCMV-IgM and HCMV-IgG among18~25 years old donors was lowest (P <0.05). Positive rate of HCMV-IgG in college students was lower than other occupation blood donars (P <0.05)and education de-gree was independent of HCMV-IgG,HCMV-IgM positive rates (P >0.05).Conclusion In this investigation,2 010 cases of voluntary blood donors from five districts of Beijing were not found in cases of HTLV infection,HCMV infection was prevalent.

Because of the specificity of infectious diseases , the the medical ethics education and concepts need to be paid more attention to .During the clinical teaching of infectious diseases , pay attention to teachers′medical ethics accomplishment level of ascension , teaching teachers should play the role of shape model , encourage interac-tion, eliminate the fear and discrimination interns and strengthen medical ethics consciousness and the combination of legal concepts , promote the medical students medical ethics consciousness of the form .

ObjectiveTo monitor the early blood lactic acid concentration of patients with severe trauma who have been experienced routine surgery or damage control surgery,and explore the influence of surgical methods for the early lactate clearance rate.MethodsSelected 40 patients with severe trauma,they were divided into two groups with 20 cases each in accordance with the adopted operation mode,reference group by damage control surgery,and control group by routine surgery.Recorded acute physiology and chronic health evaluation Ⅱ (APACHE Ⅱ ) in patients after admission,blood lactic acid concentration at 6 h after admission and admission,calculated the early lactate clearance rate.ResultsIn reference group,blood lactic acid concentration at 6 h after admission was significantly lower than that in control group[ (3.5 ± 1.1 )mmol/L vs.(4.2 ± 1.4) mmol/L,P＜ 0.05 ],early lactate clearance rate was higher than that in control group [ (24.6 ± 6.3 )％ vs.( 11.4 ± 5.3 )％,P＜ 0.05 ].Conclusions Damage control surgery in patients with severe trauma in favour of the early removal of lactic acid,maintaining the homeostasis of the organism,is the key to improve the achievement ratio of treatment with severe trauma.

Objective To study the expression of active efflux pump AdeABC,AdeIJK,AdeFGH,AbeM,AbeS,CraA,MdtL in clinical Acinetobacter baumannii isolates and whether the efflux pumps confers resistance to antibiotics.Methods Thirty-two multi-drug resistant Acinetobacter baumannii islates and 10 sensitive isolates were collected.Genes of the exporter protein were amplified by PCR.The expression of adeB,adeJ,adeG,abeM,abeS,craA,mdtL were examined by real-time fluorescence quantitative RT-PCR.The controlling genes adeRS and adeL were amplified by PCR and sequenced.Results The positivity rates of adeB,adeJ,adeG,abeM,abeS,craA,mdtL were 100％,100％,100％,96.88％,100％,100％ and 93.75％ respectively in 32 multi-drug resistant Acinetobacter baumannii isolates,and all 100％ in 10 sensitive isolates.The difference of expression of adeB,abeM and mdtL were significant( P＜0.001,P =0.001,P=0.013) between 21 multi-drug resistant isolates of C clone and 10 sensitive isolates.The mutations of adeRS existed in 2 multi-drug resistant isolates,no point mutation of adeL.Conclusion The expression of AdeABC,AbeM and MdtL may involved in the resistant mechanisms of the clinical Acinetobacter baumannii islates.

This article introduces some commonly used methods of ozone concentration detection, including chemical method, UV absorption method, and electrochemical method etc., introduces the latest four ozone concentration sensors, and summarizes the advantages and disadvantages of each method. In addition, the article emphatically introduces the ozone's applications and development in the medical aspects. Prospects for the use of ozone concentration detection, ozone treatment and ozone therapy instrument are also demonstrated in it. The literature collected and reviewed on ozone concentration detection and ozone therapy includes 37 papers in English, and 50 papers in Chinese, but only 30 articles among them are included in this review (19 in Chinese and 11 in English), according to the principle of eliminating the old information and repetitive contents. The present paper selects only those on ozone, ozone concentration, ozone therapy and ozone therapy instrument.
Hepatitis ; drug therapy ; Humans ; Mouth Diseases ; drug therapy ; Ozone ; analysis ; therapeutic use

Objective To explore the effect iveness and safety of ProSeal laryngeal mask(PLMA)used in posterior spinal surgery. Methods Forty ASA Ⅰ - Ⅱ patients of thoracic-lumbar fracture were randomly divided into PLMA group and tracheal intubation (TI) group by systematic sampling with 20 cases each. PLMA or TI was inserted after intravenous anesthesia induction. The number of intubation, intubation time and time to surgery were recorded, systolic blood pressure (SBP), diastolic blood pressure (DBP), and heart rate (HR) were recorded induction (To), during intubation (T1), at 1 min (T2),3 min(T3), 5 min (T4), 10 min after intubation (T5), during extubation (T6). The intubation, extubation, and the maintain ventilation process of the respiratory and respiratory damage were recorded. Results All patients in PLMA group and TI group achieved satisfactory lung ventilation at the first attempt. There were no significant difference in the number of intubation, intubation time and time to surgery between two groups (P ＞ 0.05 ).SBP,DBP,HR at T1,T2,T3,T6 in TI group were significandy higher than To and those in PLMA group (P＜0.05). The intubation, extubation, and the maintain ventilation process of the respiratory and respiratory damage in TI group (5,25,36 cases) were more than those in PLMA group (0,1,6 cases)(P ＜0.05).Conclusion PLMA for posterior spinal surgery is safe and effective.