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.

Objective:To retrospectively analyze and compare the clinical effects of rigid neuroendoscopic hematoma removal and drilling irrigation drainage in the treatment of chronic subdural hematomas with mixed density on head CT, and explore the appropriate surgical methods for chronic subdural hematomas with mixed density.Methods:A retrospective case-control study was conducted to analyze the clinical data of 80 patients with CSDH with mixed density CT findings admitted to the Department of Neurosurgery of Guizhou Medical University Affiliated Hospital from January 2021 to November 2023. There were 57 males and 23 females. According to the surgical method, patients were divided into endoscopic group ( n=36) and drilling group ( n=44). Patients in the endoscopic group underwent hard neuroendoscopic hematoma removal surgery, while patients in the drilling group underwent drilling flushing and drainage surgery. Compared the surgical time, drainage time, hematoma clearance rate, length of hospital stay, markwalder neurological function grading, and activities of daily living (ADL) score between two groups 30 days after surgery. Followed up for 3 months to record the recurrence situation.Measurement data with a normal distribution were expressed as mean±standard deviation ( ± s), and t-test was used for inter-group comparison. Measurement data with a non-normal distribution were expressed as M( Q1, Q3), and rank sum test was used for inter-group comparison. Chi-square test or Fisher′s exact test was used for inter-group comparison of count data. Rank sum test was used for inter-group comparison of ordinal data. Results:The operation time, postoperative drainage time, 24-hour hematoma clearance rate, midline deviation distance and hospital stay in the endoscopic group respectively were (77.50±8.15) min, 1.00(1.00, 2, 00) d, (95.00±2.66)%, 1.00(0.00, 2.00) mm, (9.47±2.52) d. The drilling group were (44.77±6.56) min, 3.00(2.25, 3.00) d, (87.86±3.43)%, 3.00(2.00, 3.00) mm, (11.84±3.28) d, the difference was statistically significant between the two groups ( P<0.05). Comparison of long-term efficacy in the endoscopic group, the hematoma clearance rate at 30 days after operation, the ADL score at 30 days after operation, and the number of recurrence cases at 3 months after operation respectively were 99.00(97.25, 100.00)%, (88.06±7.86) points, and 1 case. The drilling group were 93.50(91.25, 95.75)%, (83.29±9.58) points and 10 cases, with statistical difference between the two groups( P<0.05). 30 day postoperative Markwalder neurological functional grading, there were 27 cases grades 0, 9 cases of grade I, and 0 cases of grade II in endoscopic surgery. In the drilling group, there were 24 cases, 15 cases, and 5 cases, respectively. The difference between the two groups was statistically significant ( P<0.05). Conclusions:Compared with drilling and drainage irrigation surgery, neuroendoscopic treatment of mixed density chronic subdural hematoma takes a relatively long time, but the hematoma clearance rate is higher, the hospitalization time is shorter, the postoperative recovery is faster and the recurrence rate is lower. Neuroendoscopic therapy has unique advantages and may be more suitable for the treatment of mixed density CSDH.

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.