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.

Demyelination and remyelination play key roles in spinal cord injury (SCI), affecting the recovery of motor and sensory functions. Research in rodent models is extensive, but the study of these processes in non-human primates is limited. Therefore, our goal was to thoroughly study the histological features of demyelination and remyelination after contusion injury of the cervical spinal cord in Macaca fascicularis. In a previous study, we created an SCI model in M. fascicularis by controlling the contusion displacement. We used Eriochrome Cyanine staining, immunohistochemical analysis, and toluidine blue staining to evaluate demyelination and remyelination. The results showed demyelination ipsilateral to the injury epicenter both rostrally and caudally, the former mainly impacting sensory pathways, while the latter primarily affected motor pathways. Toluidine blue staining showed myelin loss and axonal distension at the injury site. Schwann cell-derived myelin sheaths were only found at the center, while thinner myelin sheaths from oligodendrocytes were seen at the center and surrounding areas. Our study showed that long-lasting demyelination occurs in the spinal cord of M. fascicularis after SCI, with oligodendrocytes and Schwann cells playing a significant role in myelin sheath formation at the injury site.
Animals ; Spinal Cord Injuries/physiopathology* ; Demyelinating Diseases/etiology* ; Remyelination/physiology* ; Macaca fascicularis ; Disease Models, Animal ; Myelin Sheath/pathology* ; Oligodendroglia/pathology* ; Schwann Cells/pathology* ; Female ; Spinal Cord/pathology* ; Axons/pathology*

Objective     To explore the efficacy of prone positioning ventilation in patients with acute respiratory distress syndrome (ARDS) after acute Stanford type A aortic dissection (STAAD) surgery. Methods     From November 2019 to September 2021, patients with ARDS who was placed prone position after STAAD surgery in the Xiamen Cardiovascular Hospital of Xiamen University were collected. Data such as the changes of blood gas, respiratory mechanics and hemodynamic indexes before and after prone positioning, complications and prognosis were collected for statistical analysis. Results    A total of 264 STAAD patients had surgical treatment, of whom 40 patients with postoperative ARDS were placed prone position. There were 37 males and 3 females with an average age of 49.88±11.46 years. The oxygen partial pressure, oxygenation index and peripheral blood oxygen saturation 4 hours and 12 hours after the prone positioning, and 2 hours and 6 hours after the end of the prone positioning were significantly improved compared with those before prone positioning ventilation (P<0.05). The oxygenation index 2 hours after the end of prone positioning which was less than 131.42 mm Hg, indicated that the patient might need ventilation two or more times of prone position. Conclusion     Prone position ventilation for patients with moderate to severe ARDS after STAAD surgery is a safe and effective way to improve the oxygenation.

AIM: To investigate the role of histidine-rich glycoprotein(HRG)in the neovascularization of diabetic retinopathy in rats.METHODS: Streptozocin(STZ)-induced diabetic Sprague-Dawley(SD)rats were utilized as an experimental model, the protein expression of HRG and vascular endothelial growth factor(VEGF)in the retinas of normal(Wild type, WT)and diabetic(diabetic mellitus, DM)groups was detected using Western blot(WB). The protein expression of HRG in high-glucose-induced human retinal microvascular endothelial cells(hRMECs)was verified by WB after transfection with HRG small interfering RNA(siRNA)low-expression sequences. The optimal si-HRG#298 sequence was selected for further experiments. In the animal experiment, HRG gene silencing was achieved using an adeno-associated virus(AAV)vector, with AAV2-sh-NC and AAV2-sh-HRG#298 serving as the HRG gene silencing group and the HRG empty vector control group, respectively. The protein expression of HRG and VEGF in each group was then detected by WB following the verification of HRG protein expression. Retinal structural changes were observed by HE staining, and neovascularization changes were observed by PAS staining.RESULTS: HE staining found that the retinal structure in the DM group was disordered, the number of cells in the ganglion cell layer decreased, the number of cells in the inner and outer nuclear layers decreased, and the total retinal thickness also decreased(P<0.05); cellular capillaries were significantly increased in DM rats observed by PAS staining(P<0.05); the protein expression of HRG and angiogenesis factor VEGF was up-regulated in the retina of DM group(P<0.05); the protein expression of HRG was significantly downregulated in high glucose-induced hRMECs(P<0.05); the inhibition of neovascularization in diabetic retinas and the downregulation of VEGF protein expression were achieved through HRG gene silencing(P<0.05).CONCLUSION: HRG promotes neovascularization in the retinas of diabetic rats, and HRG gene silencing can inhibit neovascularization.

OBJECTIVE To analyze the types and control measures of major microorganisms in pharmaceutical water systems, so as to provide guidance for effective control of pharmaceutical water systems. METHODS The main microbial species, abundance and harmfulness of drinking water, purified water and water for injection were reviewed, and the control measures on microorganisms in pharmaceutical water were discussed. RESULTS There were differences in the main microbial types in pharmaceutical water. Burkholderia cepacia complex and Ralstonia pickettii were conditioned pathogens in pharmaceutical water, thus causing certain biological safety hazards. CONCLUSION Pharmaceutical companies can strengthen the control of microorganisms in the water system by establishing microbial databases and common microbial strain banks at all levels. Trend analysis should to be conducted based on alert limits and action limits, so as to strengthen the control of microorganisms in the water system.

Objective:To explore the surgical strategies and clinical efficacy for aortic dissection combined with refractory superior mesenteric artery (SMA) ischemia.Methods:This is a retrospective case series study. Clinical data of 24 patients with aortic dissection and refractory SMA ischemia admitted to the Department of Vascular Surgery, Zhongshan Hospital, Fudan University from August 2010 to August 2020 were retrospectively collected. Of the 24 patients, 21 were males and 3 were females, with an age of (50.3±9.9) years (range: 44 to 72 years).Among them, 9 cases were Stanford type A aortic dissection, and 15 cases were type B. All patients underwent CT angiography upon admission, and based on imaging characteristics, they were classified into three types. Type Ⅰ: severe stenosis/occlusion of the SMA true lumen only; Type Ⅱ: stenosis of the true lumens in the descending aorta and SMA (isolated type); Type Ⅲ: stenosis of the true lumens in the thoracoabdominal aorta and SMA (continuation type). Surgical procedures, complications, mortality, and reintervention rates were recorded.Results:Among the 24 patients, 17 (70.8%) were classified as Type Ⅰ, 4 (16.7%) as Type Ⅱ, and 3 (12.5%) as Type Ⅲ. Fourteen cases of Type Ⅰ underwent thoracic endovascular aortic repair combined with SMA stent implantation. Additionally, 3 Type Ⅰ and 1 Type Ⅱ patients underwent only SMA reconstruction (with one case of chronic TAAD treated with iliac artery-SMA bypass surgery). Moreover, 3 Type Ⅱ and 3 Type Ⅲ patients underwent descending aorta combined with SMA stent implantation. There were 5 patients (20.8%) who underwent small bowel resection, either in the same sitting or in a staged procedure. During hospitalization, 4 patients died, resulting in a mortality rate of 16.7%. Among these cases, two patients succumbed to severe intestinal ischemia resulting in multiple organ dysfunction syndrome. The follow-up duration was (46±9) months (range: 13 to 72 months). During the follow-up, 2 patients died, unrelated to intestinal ischemia. The 5-year freedom from reintervention survival rate was 86.1%, and the 5-year cumulative survival rate was 82.6%.Conclusions:Patients with aortic dissection and refractory SMA ischemia have a high perioperative mortality. However, implementing appropriate surgical strategies according to different clinical scenarios can reduce mortality and alleviate intestinal ischemia.

Objective:To evaluate the clinical outcomes of thoracic endovascular aortic repair (TEVAR) in the treatment of Stanford type B aortic dissection (TBAD) in Marfan syndrome patients who had no history of aortic arch replacement.Methods:This is a retrospective case-series study. From January 2009 to December 2019,the clinical data of Marfan syndrome patients who underwent TEVAR for TBAD at the Department of Vascular Surgery were collected. A total of 23 patients were enrolled,including 15 males and 8 females. The age was (38.0±11.0) years (range:24 to 56 years). Among them,12 patients had history of ascending aortic surgery. Details of TEVAR,perioperative complications and reintervention were recorded and survival rate was analyzed by Kaplan-Meier curve.Results:Technical success was 91.3% (21/23). Two patients with technical failure were as follows:one patient had type Ⅰa endoleak at the completion angiography,which healed spontaneously during the follow-up,and the other patient suffered aortic intimal intussusception after the deployment of the first stent-graft, and the second stent-graft was deployed. However, type Ⅲ endoleak was detected,which disappeared during the follow-up. One patient died during hospitalization. The median follow-up time ( M(IQR)) was 60 (48) months (range:12 to 132 months). Reintervention was performed on 7 patients,including 3 distal stent-graft-induced new entry,2 distal aortic dilation,1 Ⅰa endoleak and 1 retrograde type A aortic dissection,respectively. Five-year cumulative survival rate was 86.7% (95% CI:86.6% to 86.8%) and the 5-year freedom from reintervention rate was 81.8% (95% CI:61.8% to 92.8%). Conclusions:TEVAR is feasible in the treatment of TBAD in Marfan syndrome patients who has no history of aortic arch replacement. It has high technical success rate and low perioperative complication.

Objective To evaluate the performance of the Quality of Life Instrument for Chronic Dis-ease Nephrotic Syndrome [QLICD-NS(V2.0)] in patients with nephrotic syndrome.Methods A total of 203 patients with nephrotic syndrome (NS) diagnosed in the Department of Nephrology,Affiliated Hospital of Guangdong Medical University from March 2021 to November 2021 were selected for QLICD-NS(V2.0) eval-uation,and the evaluation methods were generalization theory (GT) and item response theory (IRT).The dif-ficulty,discrimination coefficient and information amount of each item were obtained by using Multilog 7.0 software to analyze the grade response model (GRM) of IRT.Results The results of GT showed that the contribution ratio of the global total score in the four domains of QLICD-NS(V2.0) scale was evenly distribu-ted,and the generalization coefficient of the four domains was greater than 0.50.Except for social function,the variance component of the participants in the other three domains were greater than the item variance compo-nent,and the reliability index of each domain was greater than 0.50.The results of IRT showed that the dis-crimination degree of QLICD-NS(V2.0) scale was 0.82.Except for items TNS7 and TNS8,the difficulty coef-ficients of the other items ranged from -3 to 3 and increase monotonically.Conclusion The QLICD-NS (V2.0) scale has good reliability in physiological function and psychological function,and is acceptable in so-cial field and special function.The QLICD-NS(V2.0) scale developed in this study has good performance.

N6-methyladenosine(m6A)is produced by methylation of the 6th nitrogen atom of ade-nine.The m6A methylation modification is the most common internal modification of RNA,and the modifica-tion process mainly relies on m6A-related enzymes,including methyltransferases,demethylases and binding proteins.The core structure of the methyltransferase complex(MTC)is methyltransferase-like 3(METTL3).The pathogenesis of osteoarthritis(OA)is intricate and complex,and various factors interact with each other.This paper summarizes the mechanism of action of m6A and related enzymes,and elaborates the pathogenesis of os-teoarthritis from the aspects of chondrocytes,proteases,cytokines and signaling pathways,emphatically intro-duces the regulatory role of METTL3 on OA in m6A methylated modification.The aim is to provide new per-spectives on the pathogenesis of osteoarthritis in order to provide new ideas and approaches for the treatment of osteoarthritis.

Objective To investigate the value of 18F labeled prostate-specific membrane antigen(18F-PSMA)-1007 developing agent PET/CT(18F-PSMA-1007PET/CT)examination in the diagnostic evaluation and therapeutic decision of the newly diagnosed prostate cancer(PCa)and follow up after radical prostatecto-my(RP).Methods This study adopted the retrospective observational study method.A total of 68 patients receiving 18 F-PSMA-1007 PET/CT examination in this hospital from September 2022 to October 2023 were analyzed,including 36 cases of newly diagnosed PCa and 32 cases of biochemistry follow up failure after RP.A total of 30 items of clinical data were collected,including 8 items of basic clinical characteristics,7 items of pa-thology-related characteristics and 15 items of imaging characteristics.The patients clinical characteristics in the newly diagnosed PCa and biochemical failure after RP conducted the descriptive analysis.The Fisher exact probability method was used to analyze the differentiation of the SUVmax of primary lesions in different clini-cal subgroups[different tPSA levels at diagnosis,different mi-T stages,different Gleason scores at postopera-tive pathological puncture and different pathological types]in the newly diagnosed PCa group and the differ-entiation of recurrent lesion detection rates in different clinical subgroups(different tPSA in 18F-PSMA-1007 PET/CT examination,different pathological T stages,different lymph node invasion and different pathological Gleason scores in the biochemical failure after RP group.The Spearman correlation was adopted to test and analyze the correlation between the imaging features of positive lesions and tPSA.Results In the newly diag-nosed PCa group,there were 1 case of prostatic hyperplasia and 35 cases of PCa.SUVmax had no statistical differences among the primary lesions with different tPSA levels(P=0.81),different mi-T stages(P=0.70),different puncture Glleasonscores(P=0.20)and different pathological types(P=0.71).Moreover the tPSA value at diagnosis was positively correlated with the number of metastatic lesions(r=0.410,P=0.01).The clinical treatment decisions in 11 cases(31.43％)were changed according to the examination re-sults.In 9 cases of RP combined with lymph node dissection,the accuracy rate and concordance rate of 18F-PS-MA-1007 PET/CT and MRI in the lymph node detection rate all were 100％.I n the biochemical failure after RP group,the overall recurrent lesion detection rate was 71.88％(23/32),the operative area in situ recurrence(11 cases,34.38％)and bone metastasis(11 cases,34.38％)were most common.The differences of 18F-PS-MA-1007 PET/CT recurrent lesions detection rates had no statistical differences among the patients with dif-ferent tPSA levels(P=0.08),different pathological T stages(P=0.10),different postoperative pathological lymph node invasions(P=0.68)and different pathologic Gleason score in the 18F-PSMA-1007 PET/CT ex-amination.In the 18 F-PSMA-1007 PET/CT examination in the biochemical failure after RP,the tPSA value in the recurrent lesion was positively correlated with the number of recurrent lesions(r=0.48,P=0.01),SUVmax value in the recurrent lesion(r=0.46,P=0.01)and the SUVmean value(r=0.38,P=0.03).The clinical treatment decision in 18 cases(56.25％)was changed according to the examination results.Conclusion 18 F-PSMA-1007 PET/CT has good diagnostic value and efficiency for primary lesion and metastasis lesion of new-ly diagnosed PCa and recurrent foci of biochemical failure after RP.