Implantation of human urine stem cells promotes neural repair in spinal cord injury rats associated cadeharin-1 and integrin subunit beta 1 expression.

BACKGROUND: The aim of this study was to determine the effect of human urine-derived stem cells (HUSCs) for the treatment of spinal cord injury (SCI) and investigate associated the molecular network mechanism by using bioinformatics combined with experimental validation. METHODS: After the contusive SCI model was established, the HUSC-expressed specific antigen marker was implanted into the injury site immediately, and the Basso, Beattie and Bresnahan locomotor rating scale (BBB scale) was utilized to evaluate motor function so as to determine the effect of HUSCs for the neural repair after SCI. Then, the geneCards database was used to collect related gene targets for both HUSCs and SCI, and cross genes were merged with the findings of PubMed screen. Subsequently, protein-protein interaction (PPI) network, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment, as well as core network construction, were performed using Cytoscape software. Lastly, real-time quantitative polymerase chain reaction (PCR) and immunofluorescence were employed to validate the mRNA expression and localization of 10 hub genes, and two of the most important, designated as cadherin 1 (CDH1) and integrin subunit beta 1 (ITGB1), were identified successfully. RESULTS: The immunophenotypes of HUSCs were marked by CD90+ and CD44+ but not CD45, and flow cytometry confirmed their character. The expression rates of CD90, CD73, CD44 and CD105 in HUSCs were 99.49, 99.77, 99.82 and 99.51%, respectively, while the expression rates of CD43, CD45, CD11b and HLA-DR were 0.08, 0.30, 1.34 and 0.02%, respectively. After SCI, all rats appeared to have severe motor dysfunction, but the BBB score was increased in HUSC-transplanted rats compared with control rats at 28 days. By using bioinformatics, we obtained 6668 targets for SCI and 1095 targets for HUSCs and identified a total of 645 cross targets between HUSCs and SCI. Based on the PPI and Cytoscape analysis, CD44, ACTB, FN1, ITGB1, HSPA8, CDH1, ALB, HSP90AA1 and GAPDH were identified as possible therapeutic targets. Enrichment analysis revealed that the involved signal pathways included complement and coagulation cascades, lysosome, systemic lupus erythematosus, etc. Lastly, quantificational real-time (qRT)-PCR confirmed the mRNA differential expression of CDH1/ITGB1 after HUSC therapy, and glial fibrillary acidic protein (GFAP) immunofluorescence staining showed that the astrocyte proliferation at the injured site could be reduced significantly after HUSC treatment. CONCLUSIONS: We validated that HUSC implantation is effective for the treatment of SCI, and the underlying mechanisms associated with the multiple molecular network. Of these, CDH1 and ITGB1 may be considered as important candidate targets. Those findings therefore provided the crucial evidence for the potential use of HUSCs in SCI treatment in future clinic trials.

Molecular Network Mechanism Analysis of Urine Stem Cells Against Retinal Aging.

To investigate the effect and potential mechanism of human-derived urine stem cells (hUSCs) in inhibiting retinal aging by using experimental and bioinformatics. Retinal pigment epithelial cells cultured in vitro, which were randomly divided into normal group, aging group and supernatant of hUSCs group. Cell counting kit-8 detection, senescence-related ß-galactosidase, and Annexin V/PI staining were performed to detect cell viability, senescence, and apoptosis. Subsequently, bioinformatics methods were used to explore the underlying mechanisms, in which, targets both hUSCs and aging retina-related targets were obtained from GeneCards. Then, Gene Ontology, Kyoto Encyclopedia of Genes and Genomes enrichment analysis, and protein-protein interaction network were analysis, and the expressional level of hub gene was validated by q-PCR. Supernatant addition of hUSCs promoted markedly cellular proliferation, improved viability and inhibited senescence and apoptosis in vitro. A total of 1476 hUSCs-related targets (Relevance score > 20), 692 retinal disease-related targets, and 732 targets related to disease of aging were selected from GeneCards database, and 289 common targets of hUSCs against aging retina were confirmed through Venn analysis. Enrichment analysis demonstrated that hUSCs might exert its anti-apoptosis efficacy in multiple biological processes, including oxidative stress, inflammation and apoptosis, and core targets were associated with HIF-1, MAPK and PI3K-Akt signal. hUSCs inhibited retinal senescence by regulating multiply targets and signaling pathways, of these, HIF-1, MAPK, and PI3K may be important candidates.

Scutellarin inhibits the glioma cell proliferation by downregulating BIRC5 to promote cell apoptosis.

The expression changes of baculovirus inhibitor of apoptosis repeat-containing protein5 in brain glioma after administration of Scutellarin was detected. To explore the effort of scutellarin on anti-glioma by downregulating BIRC5.The effect of scutellarin on tumour growth and animal survival was detected by administering scutellarin to nude mice subcutaneous tumour formation and SD rats in situ tumour formation models. A significantly different gene BIRC5 was found by using the combination of TCGA databases and network pharmacology. And then qPCR was performed to detect the expression of BIRC5 in glioma tissues, cells and normal brain tissues and glial cells. CCK-8 was used to detect the IC50 of scutellarin on glioma cells. The wound healing assay, flow cytometry and MTT test were used to detect the effect of scutellarin on the apoptosis and proliferation of glioma cells. The expression of BIRC5 in glioma tissues was significantly higher than that in normal brain tissues. Scutellarin can significantly reduce tumour growth and improve animal's survival. After scutellarin was administered, the expression of BIRC5 in U251 cells was significantly reduced. And after same time, apoptosis increased and cell proliferation was inhibited. This original research showed that scutellarin can promote the apoptosis of glioma cells and inhibit the proliferation by downregulating the expression of BIRC5.

Cellular Localization and Distribution of TGF-ß1, GDNF and PDGF-BB in the Adult Primate Central Nervous System.

The available data on the localization of transforming growth factor beta1 (TGF-ß1), glial cell line-derived neurotrophic factor (GDNF), and platelet-derived growth factor-BB (PDGF-BB) in the adult primate and human central nervous system (CNS) are limited and lack comprehensive and systematic information. This study aimed to investigate the cellular localization and distribution of TGF-ß1, GDNF, and PDGF-BB in the CNS of adult rhesus macaque (Macaca mulatta). Seven adult rhesus macaques were included in the study. The protein levels of TGF-ß1, PDGF-BB, and GDNF in the cerebral cortex, cerebellum, hippocampus, and spinal cord were analyzed by western blotting. The expression and location of TGF-ß1, PDGF-BB, and GDNF in the brain and spinal cord was examined by immunohistochemistry and immunofluorescence staining, respectively. The mRNA expression of TGF-ß1, PDGF-BB, and GDNF was detected by in situ hybridization. The molecular weight of TGF-ß1, PDGF-BB, and GDNF in the homogenate of spinal cord was 25 KDa, 30 KDa, and 34 KDa, respectively. Immunolabeling revealed GDNF was ubiquitously distributed in the cerebral cortex, hippocampal formation, basal nuclei, thalamus, hypothalamus, brainstem, cerebellum, and spinal cord. TGF-ß1 was least distributed and found only in the medulla oblongata and spinal cord, and PDGF-BB expression was also limited and present only in the brainstem and spinal cord. Besides, TGF-ß1, PDGF-BB, and GDNF were localized in the astrocytes and microglia of spinal cord and hippocampus, and their expression was mainly found in the cytoplasm and primary dendrites. The mRNA of TGF-ß1, PDGF-BB, and GDNF was localized to neuronal subpopulations in the spinal cord and cerebellum. These findings suggest that TGF-ß1, GDNF and PDGF-BB may be associated with neuronal survival, neural regeneration and functional recovery in the CNS of adult rhesus macaques, providing the potential insights into the development or refinement of therapies based on these factors.

Analgesic Effects and Adverse Reactions of Lidocaine for Patient-Controlled Intravenous Analgesia on Patients Undergoing Open Hepatectomy: A Retrospective Analysis.

PURPOSE: The aim of this study was to investigate the effect of lidocaine for patient controlled intravenous analgesia (PCIA) in patients who underwent open hepatectomy. DESIGN: A retrospective analysis. METHODS: A total of 281 patients who underwent open hepatectomy from July 2018 to December 2018 were included. All patients were assigned into two groups: the lidocaine group (PCIA consisted of lidocaine, sufentanil, tramadol and granisetron) and the control group (PCIA consisted of sufentanil, tramadol and granisetron). The postoperative visual analogue scale (VAS) and complications (including respiratory depression, hypotension, nausea and vomiting, pruritus, numbness of the corners of the mouth, dizziness) between the groups were compared. FINDINGS: There were no significant differences between the characteristics, duration of surgery and anesthesia, and recovery of postoperative activity between the two groups. In the first 3 days after the operation, the postoperative VAS score of the lidocaine group was lower than that of the control group at resting state, while after activity, the postoperative VAS contrast results were completely opposite. In particularly, the resting state at 48 hours (h) (1.05 ± 1.25 vs 1.57 ± 1.54) after surgery and the activity state at 72 h (3.02 ± 1.51 vs 2.2 ± 1.66) after surgery (P < 0.05). The incidence of mouth numbness and dizziness were significantly increased in the lidocaine group (P < 0.05). CONCLUSION: The addition of lidocaine in PCIA was not beneficial to improve the pain during activities and increased the incidence of perioral numbness and dizziness.

P75 neurotrophin receptor as a therapeutic target for drug development to treat neurological diseases.

The interaction of neurotrophins with their receptors is involved in the pathogenesis and progression of various neurological diseases, including Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, spinal cord injury and acute and chronic cerebral damage. The p75 neurotrophin receptor (p75NTR) plays a pivotal role in the development of neurological dysfunctions as a result of its high expression, abnormal processing and signalling. Therefore, p75NTR represents as a vital therapeutic target for the treatment of neurodegeneration, neuropsychiatric disorders and cerebrovascular insufficiency. This review summarizes the current research progress on the p75NTR signalling in neurological deficits. We also summarize the present therapeutic approaches by genetically and pharmacologically targeting p75NTR for the attenuation of pathological changes. Based on the evolving knowledge, the role of p75NTR in the regulation of tau hyperphosphorylation, Aß metabolism, the degeneration of motor neurons and dopaminergic neurons has been discussed. Its position as a biomarker to evaluate the severity of diseases and as a druggable target for drug development has also been elucidated. Several prototype small molecule compounds were introduced to be crucial in neuronal survival and functional recovery via targeting p75NTR. These small molecule compounds represent desirable agents in attenuating neurodegeneration and cell death as they abolish activation-induced neurotoxicity of neurotrophins via modulating p75NTR signalling. More comprehensive and in-depth investigations on p75NTR-based drug development are required to shed light on effective treatment of numerous neurological disorders.

Patients with gastroenteric tumor after upper abdominal surgery were more likely to require rescue analgesia than lower abdominal surgery.

OBJECTIVES: To find out the reasons why patients still need to use rescue analgesics frequently after gastrointestinal tumor surgery under the patient-controlled intravenous analgesia (IV-PCA), and the different abdominal surgery patients using the difference of analgesics. METHODS: A total of 970 patients underwent abdominal operation for gastrointestinal tumors were included. According whether patients used dezocine frequently for rescue analgesics within 2 days after surgery, they assigned into two groups: RAN group (Patients who did not frequently use rescue analgesia, 406 cases) and RAY group (Patients who frequently used rescue analgesia, 564 cases). The data collected included patient's characteristics, postoperative visual analogue scale (VAS), nausea and vomiting (PONV), and postoperative activity recovery time. RESULTS: No differences were observed in the baseline characteristics. Compared with the RAN group, patients in the RAY group had a higher proportion of open surgery, upper abdominal surgery, VAS score at rest on the first 2 days after surgery and PONV, and a slower recovery of most postoperative activities. Under the current use of IV-PCA background, the proportion of rescue analgesics used by patients undergoing laparotomy and upper abdominal surgery was as high as 64.33% and 72.8%, respectively. Regression analysis showed that open surgery (vs laparoscopic surgery: OR: 2.288, 95% CI: 1.650-3.172) and the location of the tumor in the upper abdomen (vs lower abdominal tumor: OR: 2.738, 95% CI: 2.034-3.686) were influential factors for frequent salvage administration. CONCLUSIONS: In our patient population, with our IV-PCA prescription for postoperative pain control, patient who underwent open upper abdominal surgery required more rescue postoperative analgesia.

Analgesic effects of sufentanil in combination with flurbiprofen axetil and dexmedetomidine after open gastrointestinal tumor surgery: a retrospective study.

BACKGROUND: To investigated the effects of sufentanil in combination with flurbiprofen axetil and dexmedetomidine for patient-controlled intravenous analgesia (PCIA) on patients after open gastrointestinal tumor surgery, and compared this combination with traditional PCIA with pure opioids or epidural analgesia (PCEA). METHODS: Patients (n = 640) who underwent open gastrointestinal tumor surgery and received patient-controlled analgesia (PCA) were included. According to the type of PCA, patients were assigned to three groups: MPCIA (PCIA with sufentanil, flurbiprofen axetil, dexmedetomidine and metoclopramide), OPCIA (PCIA with sufentanil, tramadol and metoclopramide) and PCEA group (PCEA with sufentanil and ropivacaine). The characteristics of patients, intraoperative use of analgesics, postoperative visual analogue scale (VAS), postoperative adverse reactions and postoperative recovery were collected. The primary outcome was postoperative VAS score. One-way ANOVA, Kruskal-Wallis H test, Fisher exact probability method, and binary logistic regression analysis were used for analysis. RESULTS: There were no significant differences in the characteristics of patients, operation time, tumor site and the use of postoperative rescue analgesics among the groups. In the first two days after open gastrointestinal tumor surgery, the VAS (expressed by median and interquartile range) of MPCIA (24th h, resting: 1,1; movement: 3,2. 48th h, resting: 0,1; movement: 2,1.) and PCEA (24th h, resting: 0,1; movement: 2,1. 48th h, resting: 0,1; movement: 2,2.) groups were significantly lower than those of OPCIA group (24th h, resting: 2.5,2; movement: 4,2. 48th h, resting: 1.5,1.75; movement: 3,1.) (all p <  0.01). The incidence of postoperative nausea and vomiting in MPCIA group was 13.6% on the first day after surgery, which was significantly higher than that in PCEA group. There was no significant difference in the incidence of other postoperative adverse events. Higher intraoperative sufentanil dosage (OR (95%CI) = 1.017 (1.002-1.031), p = 0.021), lower body mass index (OR (95%CI) = 2.081 (1.059-4.089), p = 0.033), and tumor location above duodenum (OR (95%CI) = 2.280 (1.445-3.596), p <  0.001) were associated with poor postoperative analgesia. CONCLUSIONS: The analgesic effects of PCIA with sufentanil in combination with flurbiprofen axetil and dexmedetomidine on postoperative analgesia was better than that of traditional pure opioids PCIA, and similar with that of PCEA.

Reduced Expression of Voltage-Gated Sodium Channel Beta 2 Restores Neuronal Injury and Improves Cognitive Dysfunction Induced by Aß1-42.

Voltage-gated sodium channel beta 2 (Nav2.2 or Navß2, coded by SCN2B mRNA), a gene involved in maintaining normal physiological functions of the prefrontal cortex and hippocampus, might be associated with prefrontal cortex aging and memory decline. This study investigated the effects of Navß2 in amyloid-ß 1-42- (Aß1-42-) induced neural injury model and the potential underlying molecular mechanism. The results showed that Navß2 knockdown restored neuronal viability of Aß1-42-induced injury in neurons; increased the contents of brain-derived neurotrophic factor (BDNF), enzyme neprilysin (NEP) protein, and NEP enzyme activity; and effectively altered the proportions of the amyloid precursor protein (APP) metabolites including Aß42, sAPPα, and sAPPß, thus ameliorating cognitive dysfunction. This may be achieved through regulating NEP transcription and APP metabolism, accelerating Aß degradation, alleviating neuronal impairment, and regulating BDNF-related signal pathways to repair neuronal synaptic efficiency. This study provides novel evidence indicating that Navß2 plays crucial roles in the repair of neuronal injury induced by Aß1-42 both in vivo and in vitro.

Panax notoginseng saponin attenuates the hypoxic-ischaemic injury in neonatal rats by regulating the expression of neurotrophin factors.

Neonatal hypoxic-ischaemic (HI) injury is a serious complication of neonatal asphyxia and the leading cause of neonatal acute death and chronic neurological injury, and the effective therapeutic method is lacking to improve patients' outcomes. We reported in this study that panax notoginseng saponin (PNS) may provide a treatment option for HI. HI model was established using neonatal Sprague-Dawley rats and then intraperitoneally injected with different dosage of PNS, once a day for 7 days. Histological staining and behavioural evaluations were performed to elucidate the pathological changes and neurobehavioural variation after PNS treatment. We found PNS administration significantly reduced the infarct volume of brain tissues and improved the autonomous activities of neonatal rats, especially with higher dosage. PNS treatment at 40 mg/kg reduced neuronal damage, suppressed neuronal apoptosis and depressed astroglial reactive response. Moreover, the long-term cognitive and motor functions were also improved after PNS treatment at 40 mg/kg. Importantly, PNS treatment elevated the levels of BDNF and TrkB but decreased the expression of p75NTR both in the cortex and hippocampus of HI rats. The therapeutic efficacy of PNS might be correlated with PNS-activated BDNF/TrkB signalling and inactivation of p75NTR expression, providing a novel potential therapy for alleviating HI injury.

The efficacy of ultrasound-guided stellate ganglion block in alleviating postoperative pain and ventricular arrhythmias and its application prospects.

Stellate ganglion block (SGB) has been applied in clinic for almost a century as a therapeutic procedure to alleviate pain-related syndromes and vascular deficits in the upper extremities. A great number of causative side effects and complications due to technological insufficiency and anatomical variations called for the popularity of ultrasound-guided SGB which has made tremendous contribution for clinical diagnosis and therapy, primarily in postoperative pain and cardiac and vascular disorders. This work was aimed at systematically summarizing the current clinical application of ultrasound-guided SGB and putting forward the potential prospective application in future. By searching ultrasound-guided SGB-related works on PubMed database, we mainly elucidated the analgesic effect of preoperative SGB in patients undergoing surgical procedures and substantial reduction in patients with ventricular arrhythmias. The volume of local anesthetics used in ultrasound-guided SGB has been diminished in the recent few years' investigations and successful operation of ultrasound-guided SGB could be achieved with minimal safe volume of local anesthetics. This invasive and safe procedure shows vast potential for future development in clinical treatment for autonomic nervous system and autoimmune disorders. We also put forward hypothesis that ultrasound-guided SGB could be applied combined with controlled hypotension to reduce the intraoperative complications in orthopedic surgery such as insufficiency of cerebral blood flow and reflexive tachycardia. Thus, it is of vital essence to improve the professional skills of physicians for the high rate of success and explore more effective measures which could enhance therapeutic effects when combined with ultrasound-guided SGB in alleviating misery of patients.

Complexin I knockout rats exhibit a complex neurobehavioral phenotype including profound ataxia and marked deficits in lifespan.

Complexin I (CPLX1), a presynaptic small molecule protein, forms SNARE complex in the central nervous system involved in the anchoring, pre-excitation, and fusion of axonal end vesicles. Abnormal expression of CPLX1 occurs in several neurodegenerative and psychiatric disorders that exhibit disrupted neurobehaviors. CPLX1 gene knockout induces severe ataxia and social behavioral deficits in mice, which has been poorly demonstrated. Here, to address the limitations of single-species models and to provide translational insights relevant to human diseases, we used CPLX1 knockout rats to further explore the function of the CPLX1 gene. The CRISPR/Cas9 gene editing system was adopted to generate CPLX1 knockout rats (CPLX1-/-). Then, we characterized the survival rate and behavioral phenotype of CPLX1-/- rats using behavioral analysis. To further explain this phenomenon, we performed blood glucose testing, Nissl staining, hematoxylin-eosin staining, and Golgi staining. We found that CPLX1-/- rats showed profound ataxia, dystonia, movement and exploratory deficits, and increased anxiety and sensory deficits but had normal cognitive function. Nevertheless, CPLX1-/- rats could swim without training. The abnormal histomorphology of the stomach and intestine were related to decreased weight and early death in these rats. Decreased dendritic branching was also found in spinal motor neurons in CPLX1-/- rats. In conclusion, CPLX1 gene knockout induced the abnormal histomorphology of the stomach and intestine and decreased dendritic branching in spinal motor neurons, causing different phenotypes between CPLX1-/- rats and mice, even though both of these phenotypes showed profound ataxia. These findings provide a new perspective for understanding the role of CPLX1.

COX5A over-expression protects cortical neurons from hypoxic ischemic injury in neonatal rats associated with TPI up-regulation.

BACKGROUND: Neonatal hypoxic-ischemic encephalopathy (HIE) represents as a major cause of neonatal morbidity and mortality. However, the underlying molecular mechanisms in brain damage are still not fully elucidated. This study was conducted to determine the specific potential molecular mechanism in the hypoxic-ischemic induced cerebral injury. METHODS: Here, hypoxic-ischemic (HI) animal models were established and primary cortical neurons were subjected to oxygen-glucose deprivation (OGD) to mimic HIE model in vivo and in vitro. The HI-induced neurological injury was evaluated by Zea-longa scores, Triphenyte-trazoliumchloride (TTC) staining the Terminal Deoxynucleotidyl Transferased Utp Nick End Labeling (TUNEL) and immunofluorescent staining. Then the expression of Cytochrome c oxidase subunit 5a (COX5A) was determined by immunohistochemistry, western blotting (WB) and quantitative real time Polymerase Chain Reaction (qRT-PCR) techniques. Moreover, HSV-mediated COX5A over-expression virus was transducted into OGD neurons to explore the role of COX5A in vitro, and the underlying mechanism was predicted by GeneMANIA, then verified by WB and qRT-PCR. RESULTS: HI induced a severe neurological dysfunction, brain infarction, and cell apoptosis as well as obvious neuron loss in neonatal rats, in corresponding to the decrease on the expression of COX5A in both sides of the brain. What's more, COX5A over-expression significantly promoted the neuronal survival, reduced the apoptosis rate, and markedly increased the neurites length after OGD. Moreover, Triosephosephate isomerase (TPI) was predicted as physical interactions with COX5A, and COX5A over-expression largely increased the expressional level of TPI. CONCLUSIONS: The present findings suggest that COX5A plays an important role in promoting neurological recovery after HI, and this process is related to TPI up-regulation.

[Evaluating Cardiac Function of Tree Shrew (Tupaiabelangeri) using CMR at 7T with 2D Tissue Tracking].

OBJECTIVE: To determine the potential value of the two-dimensional (2D) cardiac magnetic resonance imaging (CMR) tissue tracking (CMR-TT) method in assessing the cardiac function of tree shrew at 7T. METHODS: Healthy adult tree shrews (male, n=8) and spraguedawley rats(male, n=8) were selected for this study. CMR was performed to acquire the short-axis images of left ventricle at 7T using the same appropriative coil and cine sequence for all experimental animals. The CMR images were processed using the professional cardiac analysis software, calculating ejection fraction (EF), radial peak sysolic strain (Err), circumferential peak sysolic strain (Ecc), radial peak sysolic displacement (DR), and LVM/BM 〔the ratio of left ventricular mass (LVM) to body mass (BM)〕. RESULTS: Cine imaging for the tree shrews was 100% successful following the CMR protocol for the rats, with clearly visible main segments of cardiac. Significant differences in EF, Err, Ecc and DR were found between the two groups of animals (P < 0.01). The tree shrews has lower EF, Err and Ecc than the rats. Err and Ecc appeared in the fifteenth phase in left ventriclar systole in the tree shrews, compared with the tenth phase in the rats.The tree shrews also had higher LVM/BM than the rats. CONCLUSION: The cardiac function of tree shrew can be assessed using the 2D CMR-TT method despite significant differences across species.

Effects of Alpha-Synuclein on Primary Spinal Cord Neurons Associated with Apoptosis and CNTF Expression.

Spinal cord injury (SCI) often causes neurological deficits with poor recovery; the treatment, however, is far from satisfaction, and the mechanisms remain unclear. Using immunohistochemistry and western blotting analysis, we found α-synuclein (SNCA) was significantly up-regulated in the spinal caudal segment of rats subjected to spinal cord transection at 3 days post-operation. Moreover, the role of SNCA on neuronal growth and apoptosis in vitro was determined by using overexpressing and interfering SNCA recombined plasmid vectors, and the underlying mechanism was detected by QRT-PCR and western blotting. Spinal neurons transfected with SNCA-shRNA lentivirus gave rise to an optimal neuronal survival, while it results in cell apoptosis in SNCA-ORF group. In molecular level, SNCA silence induced the up-regulation of CNTF and down-regulation of Caspase7/9. Together, endogenous SNCA plays a crucial role in spinal neuronal survival, in which the underlying mechanism may be linked to the regulation both apoptotic genes (Caspase7/9) and CNTF. The present findings therefore provide novel insights into the role of SNCA in spinal cord and associated mechanism, which may provide novel cue for the treatment of SCI in future clinic trials.

[The Improvement in the Primary Culture of Alveolar Epithelial Type of Rats].

OBJECTIVES: To improve the method of culturing and obtain purified alveolar epithelial type1 (AT1) cells of SD rats. METHODS: 1 d newborn SD rats were applied for cell culture and brains were decapitated for lung tissues obtaining after respiratory cessation. Collagenase1and DNase1 were used to digest and isolate cells. Then, cells were put into the plate coated with type1 rat tail collagen and different adherence was used to purify cells. Meanwhile, culture medium replacement was conducted after 48 h. The growth status was observed under an inverted microscope. Immunofluorescence including specific marker AQP5, SPC, BSI, Vimentin were used to identify cells. RESULTS: 2 d after incubation, the cells began to adhere to plate. At day 4 and 6, cells began to proliferate and exhibited a shape of spindle, cube and polygon. 8 d after incubation, the character of proliferation reached the highest and the cell viability was (87±8)% and purification was (87±5)%. CONCLUSION: By modifying the methods of tissues harvest, isolation and culture, we can obtain AT 1 cells with high yield, good growth state and super purity.

Injection of Aß1-40 into hippocampus induced cognitive lesion associated with neuronal apoptosis and multiple gene expressions in the tree shrew.

Alzheimer's disease (AD) can incur significant health care costs to the patient, their families, and society; furthermore, effective treatments are limited, as the mechanisms of AD are not fully understood. This study utilized twelve adult male tree shrews (TS), which were randomly divided into PBS and amyloidbetapeptide1-40 (Aß1-40) groups. AD model was established via an intracerebroventricular (icv) injection of Aß1-40 after being incubated for 4 days at 37 °C. Behavioral, pathophysiological and molecular changes were evaluated by hippocampal-dependent tasks, magnetic resonance imaging (MRI), silver staining, hematoxylin-eosin (HE) staining, TUNEL assay and gene sequencing, respectively. At 4 weeks post-injection, as compared with the PBS group, in Aß1-40 injected animals: cognitive impairments happened, and the hippocampus had atrophied indicated by MRI findings; meanwhile, HE staining showed the cells of the CA3 and DG were significantly thinner and smaller. The average number of cells in the DG, but not the CA3, was also significantly reduced; furthermore, silver staining revealed neurotic plaques and neurofibrillary tangles (NFTs) in the hippocampi; TUNEL assay showed many cells exhibited apoptosis, which was associated with downregulated BCL-2/BCL-XL-associated death promoter (Bad), inhibitor of apoptosis protein (IAP), Cytochrome c (CytC) and upregulated tumor necrosis factor receptor 1 (TNF-R1); lastly, gene sequencing reported a total of 924 mobilized genes, among which 13 of the downregulated and 19 of the upregulated genes were common to the AD pathway. The present study not only established AD models in TS, but also reported on the underlying mechanism involved in neuronal apoptosis associated with multiple gene expression.

Knockdown of α-synuclein in cerebral cortex improves neural behavior associated with apoptotic inhibition and neurotrophin expression in spinal cord transected rats.

Spinal cord injury (SCI) often causes severe functional impairment with poor recovery. The treatment, however, is far from satisfaction, and the mechanisms remain unclear. By using proteomics and western blot, we found spinal cord transection (SCT) resulted in a significant down-regulation of α-synuclein (SNCA) in the motor cortex of SCT rats at 3 days post-operation. In order to detect the role of SNCA, we used SNCA-ORF/shRNA lentivirus to upregulate or knockdown SNCA expression. In vivo, SNCA-shRNA lentivirus injection into the cerebral cortex motor area not only inhibited SNCA expression, but also significantly enhanced neurons' survival, and attenuated neuronal apoptosis, as well as promoted motor and sensory function recovery in hind limbs. While, overexpression SNCA exhibited the opposite effects. In vitro, cortical neurons transfected with SNCA-shRNA lentivirus gave rise to an optimal neuronal survival and neurite outgrowth, while it was accompanied by reverse efficiency in SNCA-ORF group. In molecular level, SNCA silence induced the upregulation of Bcl-2 and the downregulation of Bax, and the expression of NGF, BDNF and NT3 was substantially upregulated in cortical neurons. Together, endogenous SNCA play a crucial role in motor and sensory function regulation, in which, the underlying mechanism may be linked to the regulation of apoptosis associated with apoptotic gene (Bax, Bcl2) and neurotrophic factors expression (NGF, BDNF and NT3). These finds provide novel insights to understand the role of SNCA in cerebral cortex after SCT, and it may be as a novel treatment target for SCI repair in future clinic trials.

Bone Marrow Stromal Cells Promote Neuronal Restoration in Rats with Traumatic Brain Injury: Involvement of GDNF Regulating BAD and BAX Signaling.

BACKGROUND/AIMS: To investigate the effects of bone marrow stromal cells (BMSCs) and underlying mechanisms in traumatic brain injury (TBI). METHODS: Cultured BMSCs from green fluorescent protein-transgenic mice were isolated and confirmed. Cultured BMSCs were immediately transplanted into the regions surrounding the injured-brain site to test their function in rat models of TBI. Neurological function was evaluated by a modified neurological severity score on the day before, and on days 7 and 14 after transplantation. After 2 weeks of BMSC transplantation, the brain tissue was harvested and analyzed by microarray assay. And the coronal brain sections were determined by immunohistochemistry with mouse anti-growth-associated protein-43 kDa (anti-GAP-43) and anti-synaptophysin to test the effects of transplanted cells on the axonal regeneration in the host brain. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay and Western blot were used to detect the apoptosis and expression of BAX and BAD. RESULTS: Microarray analysis showed that BMSCs expressed growth factors such as glial cell-line derived neurotrophic factor (GDNF). The cells migrated around the injury sites in rats with TBI. BMSC grafts resulted in an increased number of GAP-43-immunopositive fibers and synaptophysin-positive varicosity, with suppressed apoptosis. Furthermore, BMSC transplantation significantly downregulated the expression of BAX and BAD signaling. Moreover, cultured BMSC transplantation significantly improved rat neurological function and survival. CONCLUSION: Transplanted BMSCs could survive and improve neuronal behavior in rats with TBI. Mechanisms of neuroprotection and regeneration were involved, which could be associated with the GDNF regulating the apoptosis signals through BAX and BAD.

BDNF Overexpression Exhibited Bilateral Effect on Neural Behavior in SCT Mice Associated with AKT Signal Pathway.

Spinal cord injury (SCI), a severe health problem in worldwide, was commonly associated with functional disability and reduced quality of life. As the expression of brain-derived neurotrophic factor (BDNF) was substantial event in injured spinal cord, we hypothesized whether BDNF-overexpression could be in favor of the recovery of both sensory function and hindlimb function after SCI. By using BDNF-overexpression transgene mice [CMV-BDNF 26 (CB26) mice] we assessed the role of BDNF on the recovery of neurological behavior in spinal cord transection (SCT) model. BMS score and tail-flick test was performed to evaluate locomotor function and sensory function, respectively. Immunohistochemistry was employed to detect the location and the expression of BDNF, NeuN, 5-HT, GAP-43, GFAP as well as CGRP, and the level of p-AKT and AKT were examined through western blot analysis. BDNF overexpressing resulted in significant locomotor functional recovery from 21 to 28 days after SCT, compared with wild type (WT)+SCT group. Meanwhile, the NeuN, 5-HT and GAP-43 positive cells were markedly increased in ventral horn in BDNF overexpression animals, compared with WT mice with SCT. Moreover, the crucial molecular signal, p-AKT/AKT has been largely up-regulated, which is consistent with the improvement of locomotor function. However, in this study, thermal hyperpathia encountered in sham (CB26) group and WT+SCT mice and further aggravated in CB26 mice after SCT. Also, following SCT, the significant augment of positive-GFAP astrocytes and CGRP fibers were found in WT+SCT mice, and further increase was seen in BDNF over-expression transgene mice. BDNF-overexpression may not only facilitate the recovery of locomotor function via AKT pathway, but also contributed simultaneously to thermal hyperalgesia after SCT.