Objective:To discuss the differential effects of apolipoprotein E(APOE)gene polymorphism in the neurotoxicity-reactive astrocytes,and to provide the theoretical basis for the study of the pathogenesis of Alzheimer's disease(AD).Methods:The primary cortical astrocytes from the APOE-knockout mice(APOE-/-)were isolated and cultured in vitro,and the purity of the cells was identified by immunofluorescence staining.The human APOE3 and APOE4 recombinant over-expression plasmids were constructed and separately transfected into the primary APOE-/-astrocytes,and the APOE-/-primary cells were regarded as control.Western blotting method was used to detect the expression levels of APOE and glial fibrillary acidic protein(GFAP)proteins in the cells;enzyme-linked immunosorbent assay(ELISA)method was used to detect the APOE level in the cellular culture supernatant.The inflammatory models were prepared with the primary astrocytes transfected with APOE3 and APOE4 and co-stimulated with interleukin-1α(IL-1α),tumor necrosis factor(TNF),and complement C1q.The cells were divided into APOE3+PBS group,APOE4+PBS group,APOE3+IL-1α+TNF+ C1q group,and APOE4+IL-1α+TNF+C1q group.Cell immunofluorescence staining method was used to observe the morphology of the cells in various groups;real-time fluorescence quantitative PCR(RT-qPCR)method was used to detect the expression levels of glypican 4(Gpc4),glypican 6(Gpc6),thrombospondin 1(Thbs1),thrombospondin 2(Thbs2),SPARC-like protein 1(Sparcl1)and glial cell line derived neurotrophic factor(GDNF),C3,and S100 calcium binding protein B(S100B)mRNA in the cells in various groups;microsphere phagocytosis assay was used to detect the phagocytic capacities of the cells in various groups;Western blotting was used to detect the protein expression levels of B-cell lymphoma 2(Bcl-2),and cysteinyl aspartate specific protease-3(Caspase-3)proteins in the cells in various groups.Results:Compared with APOE-/-group,the expression levels of APOE and GFAP proteins in the cells and the APOE level in the cellular culture supernatant in transfected APOE3 and transfected APOE4 groups were increased(P<0.01).The fluorescence microscope observation results showed that compared with APOE3+PBS and APOE4+PBS groups,the astrocytic processes in APOE3+IL-1α +TNF+Cq1 group and APOE4+IL-1α+TNF+Cq1 group became shorter and the cell bodies became larger;compared with APOE3+IL-1α +TNF+Cq1 group,the astrocytic processes in APOE4+IL-1α +TNF+Cq1 group were even shorter.Compared with APOE3+PBS and APOE4+PBS groups,the expression levels of Gpc4,Gpc6,Thbs1,Thbs2,and Sparcl1 mRNA in the cells in APOE3+IL-1α +TNF+Cq1 group and APOE4+IL-1α +TNF+Cq1 group were significantly decreased(P<0.01);compared with APOE3+IL-1α +TNF+Cq1 group,the expression levels of Gpc4,Gpc6,Thbs1,Thbs2,and Sparcl1 mRNA in the cells in APOE4+IL-1α +TNF+Cq1 group were significantly decreased(P<0.05 or P<0.01).Compared with APOE3+PBS and APOE4+PBS groups,the expression levels of GDNF mRNA in the cells in APOE3+IL-1α+TNF+Cq1 group and APOE4+ IL-1α +TNF+Cq1 group were decreased(P<0.01),and the expression levels of C3 and S100B mRNA were increased(P<0.01);compared with APOE3+IL-1α +TNF+Cq1 group,the expression level of GDNF mRNA in the cells in APOE4+IL-1α+TNF+Cq1 group was decreased(P<0.05),and the expression levels of C3 and S100B mRNA were increased(P<0.05).Compared with APOE3+ PBS group and APOE4+PBS group,the numbers of hagocytosis of microspheres in the cells in APOE3+ IL-1α +TNF+Cq1 group and APOE4+IL-1α +TNF+Cq1 group were significantly decreased;compared with APOE3+IL-1α+TNF+Cq1 group,the number of hagocytosis of microspheres in the cells in APOE4+IL-1α+TNF+Cq1 group was significantly decreased.Compared with APOE3+PBS group and APOE4+PBS group,the expression levels of Bcl-2 protein in the cells in APOE3+IL-1α+TNF+ Cq1 group and APOE4+IL-1α +TNF+Cq1 group were decreased(P<0.05 or P<0.01)and the expression levels of Caspase-3 protein were significantly increased(P<0.01);compared with APOE3+ IL-1α+TNF+Cq1 group,the expression level of Bcl-2 protein in the cells in APOE4+IL-1α+TNF+ Cq1 group was decreased(P<0.01),and the expression level of Caspase-3 protein was increased(P<0.05).Conclusion:The APOE4 genotype has a stronger ability to induce the inflammatory factors compared with APOE3;it can lead to a neurotoxicity-reactive astrocyte phenotype,increase the neurotoxicity,affect the astrocyte apoptosis,and aggravate the neuron damage.

Objective:To evaluate the efficacy and safety of fronto-orbital plasty using premature frontal suture osteotomy with frontoparietal transposition for the treatment of metopic synostosis.Methods:Retrospective analysis was performed on the clinical data of children with metopic synostosis who were treated in the Department of Burns and Plastic Surgery, Children’ s Hospital of Nanjing Medical University from January 2015 to October 2023 with fronto-orbital plasty, which involved premature frontal suture osteotomy with frontoparietal transposition. The frontal and parietal bones were divided into two bones, then the supraorbital bridge was cut off and the premature frontal suture was removed. The parietal bone block was then placed on the upper edge of the orbital bone, followed by the placement of the two pieces of the frontal bone on the upper edge of the parietal bone. Finally, all bone blocks were secured using absorbable plates. Pre-and post-operative CT scans and reconstructions were performed to measure changes in cranial cavity volume, and complications such as cerebrospinal fluid leakage, infection, and recurrence of metopic synostosis were followed up.Results:A total of 5 patients with metopic synostosis were enrolled, 4 males and 1 female, aged 5 to 14 months. The operation time was 4.5 to 5.5 hours, and the intraoperative blood loss was 50 to 150 ml. All patients completed the treatment without serious complications. The cranial cavity volume increased by 48.53 to 172.00 cm 3 after the operation. Follow-up was conducted for 3 to 6 months, and the trigonocephaly was completely improved without any serious complications. Short-term follow-up indicated stable craniofacial morphology. Conclusion:Fronto-orbital plasty using premature frontal suture osteotomy with frontoparietal transposition is an effective and safe treatment for metopic synostosis. The procedure has a low risk of recurrence and complications, and the result are positive. Recurrence was scarce in the short term, and the long-term situation needs to be observed.

Objective:To evaluate the efficacy and safety of fronto-orbital plasty using premature frontal suture osteotomy with frontoparietal transposition for the treatment of metopic synostosis.Methods:Retrospective analysis was performed on the clinical data of children with metopic synostosis who were treated in the Department of Burns and Plastic Surgery, Children’ s Hospital of Nanjing Medical University from January 2015 to October 2023 with fronto-orbital plasty, which involved premature frontal suture osteotomy with frontoparietal transposition. The frontal and parietal bones were divided into two bones, then the supraorbital bridge was cut off and the premature frontal suture was removed. The parietal bone block was then placed on the upper edge of the orbital bone, followed by the placement of the two pieces of the frontal bone on the upper edge of the parietal bone. Finally, all bone blocks were secured using absorbable plates. Pre-and post-operative CT scans and reconstructions were performed to measure changes in cranial cavity volume, and complications such as cerebrospinal fluid leakage, infection, and recurrence of metopic synostosis were followed up.Results:A total of 5 patients with metopic synostosis were enrolled, 4 males and 1 female, aged 5 to 14 months. The operation time was 4.5 to 5.5 hours, and the intraoperative blood loss was 50 to 150 ml. All patients completed the treatment without serious complications. The cranial cavity volume increased by 48.53 to 172.00 cm 3 after the operation. Follow-up was conducted for 3 to 6 months, and the trigonocephaly was completely improved without any serious complications. Short-term follow-up indicated stable craniofacial morphology. Conclusion:Fronto-orbital plasty using premature frontal suture osteotomy with frontoparietal transposition is an effective and safe treatment for metopic synostosis. The procedure has a low risk of recurrence and complications, and the result are positive. Recurrence was scarce in the short term, and the long-term situation needs to be observed.

Objective To investigate the risk factors for bronchopulmonary dysplasia(BPD)in twin preterm infants with a gestational age of<34 weeks,and to provide a basis for early identification of BPD in twin preterm infants in clinical practice.Methods A retrospective analysis was performed for the twin preterm infants with a gestational age of<34 weeks who were admitted to 22 hospitals nationwide from January 2018 to December 2020.According to their conditions,they were divided into group A(both twins had BPD),group B(only one twin had BPD),and group C(neither twin had BPD).The risk factors for BPD in twin preterm infants were analyzed.Further analysis was conducted on group B to investigate the postnatal risk factors for BPD within twins.Results A total of 904 pairs of twins with a gestational age of<34 weeks were included in this study.The multivariate logistic regression analysis showed that compared with group C,birth weight discordance of>25%between the twins was an independent risk factor for BPD in one of the twins(OR=3.370,95%CI:1.500-7.568,P<0.05),and high gestational age at birth was a protective factor against BPD(P<0.05).The conditional logistic regression analysis of group B showed that small-for-gestational-age(SGA)birth was an independent risk factor for BPD in individual twins(OR=5.017,95%CI:1.040-24.190,P<0.05).Conclusions The development of BPD in twin preterm infants is associated with gestational age,birth weight discordance between the twins,and SGA birth.

AIM: To investigate the effect of repeated intravitreal injection of ranibizumab and aflibercept on corneal nerve of patients with macular edema.METHODS: A total of 64 patients(64 eyes)enrolled in our hospital from June 2021 to June 2022 were treated with intravitreal injection of anti-vascular endothelial growth factor(VEGF). There were 20 cases(20 eyes)of diabetic macular edema, 19 cases(19 eyes)of wet age-related macular degeneration and 25 cases(25 eyes)of retinal vein occlusion. Corneal confocal microscope was used to collect images of corneal subbasal nerve plexus before injections and at 1mo after each intravitreal injection based on 3+pro re nata(PRN)treatment regimen. Furthermore, the length and density of corneal nerve were measured.RESULTS: There was no significant difference in corneal nerve density of patients injected with aflibercept between pre-injection and post-injection(P>0.05), while the corneal nerve length after 2nd and 3rd injections was lower than that of pre-injection(all P<0.01). There were no significant changes in corneal nerve density and length in patients with intravitreal injections of ranibizumab(all P>0.05), and there was no significant differences in corneal nerve density and length after 3 injections of the two drugs(all P>0.05).CONCLUSION: Repeated intravitreal anti-VEGF drug may affect corneal nerve to some extent. For patients who need repeated intravitreal injections of anti-VEGF, attention should be paid to the changes of corneal nerves.

Objective: To explore a simple and feasible method for the isolation and purification of hepatocytes, hepatic stellate cells (HSC), and lymphocytes from mice. Methods: The cell suspension was obtained from male C57bl/6 mice by hepatic perfusion through the portal vein digestion method and then isolated and purified by discontinuous Percoll gradient centrifugation. Trypan blue exclusion was used to determine cell viability. Glycogen staining, cytokeratin 18, and transmission electron microscopy were used to identify hepatic cells. Immunofluorescence was used to detect α-smooth muscle actin combined with desmin in HSCs. Flow cytometry was used to analyze lymphocyte subsets in the liver. Results: After isolation and purification, about 2.7×10(7) hepatocytes, 5.7×10(5) HSCS, and 4.6×106 hepatic mononuclear cells were obtained from the liver of mice with a body weight of about 22g. The cell survival rate in each group was > 95%. Hepatocytes were apparent in glycogen deposited purple-red granules and cytokeratin 18. Electron microscopy showed that there were abundant organelles in hepatocytes and tight junctions between cells. HSC had expressed α-smooth muscle actin and desmin. Flow cytometry showed hepatic mononuclear cells, including lymphocyte subsets such as CD4, CD8, NKs, and NKTs. Conclusion: The hepatic perfusion through the portal vein digestion method can isolate multiple primary cells from the liver of mice at once and has the features of simplicity and efficiency.
Male ; Mice ; Animals ; Keratin-18 ; Actins ; Desmin ; Liver ; Hepatocytes ; Hepatic Stellate Cells

Humans ; Child ; Nasal Bone ; Neurilemmoma/surgery*

Objective To investigate the difference of matrix stiffness in different regions of tibial plateau in osteoarthritis (OA) and its effects on morphology of the cartilage and mitochondria. Methods The tibial plateau cartilage specimens of OA were obtained for nanoindentation test, transmission electron microscopy and histological analysis. The stiffness of cartilage matrix in different regions of OA tibial plateau was detected by nano-indentation. The morphology of cartilage mitochondria in different regions was observed by transmission electron microscopy, and the changes of mitochondrial plane area, shape and ridge volume density were quantitatively analyzed. Cartilage injury in different regions of OA tibial plateau was observed by histological staining. Results The cartilage of OA tibial plateau showed regional heterogeneity, and the cartilage and mitochondria on medial side of varus knee OA were more severe, and the matrix stiffness was higher. The OA scores were positively correlated with matrix stiffness. There was also a significant correlation between OA scores and mitochondrial morphology: the higher OA scores, the larger and rounder mitochondrial plane area, and the lower cristae volume density. Conclusions The differences of tibial plateau revealed the correlation between cartilage matrix stiffness, OA scores and mitochondrial morphological parameters. The increased cartilage matrix stiffness may be the main cause of chondrocyte mitochondrial injury, and further aggravate the progression of OA.

The development of chemoresistance which results in a poor prognosis often renders current treatments for colorectal cancer(CRC).In this study,we identified reduced microvessel density(MVD)and vascular immaturity resulting from endothelial apoptosis as therapeutic targets for overcoming chemoresistance.We focused on the effect of metformin on MVD,vascular maturity,and endothelial apoptosis of CRCs with a non-angiogenic phenotype,and further investigated its effect in overcoming chemoresistance.In situ transplanted cancer models were established to compare MVD,endothelial apoptosis and vascular maturity,and function in tumors from metformin-and vehicle-treated mice.An in vitro co-culture system was used to observe the effects of metformin on tumor cell-induced endothelial apoptosis.Transcriptome sequencing was performed for genetic screening.Non-angiogenic CRC developed inde-pendently of angiogenesis and was characterized by vascular leakage,immaturity,reduced MVD,and non-hypoxia.This phenomenon had also been observed in human CRC.Furthermore,non-angiogenic CRCs showed a worse response to chemotherapeutic drugs in vivo than in vitro.By suppressing endo-thelial apoptosis,metformin sensitized non-angiogenic CRCs to chemo-drugs via elevation of MVD and improvement of vascular maturity.Further results showed that endothelial apoptosis was induced by tumor cells via activation of caspase signaling,which was abrogated by metformin administration.These findings provide pre-clinical evidence for the involvement of endothelial apoptosis and subsequent vascular immaturity in the chemoresistance of non-angiogenic CRC.By suppressing endothelial apoptosis,metformin restores vascular maturity and function and sensitizes CRC to chemotherapeutic drugs via a vascular mechanism.