Upregulated DNA Damage-Linked Biomarkers in Parkinson's Disease Model Mice.

SUMMARY STATEMENT: The present study examined expression of DNA damage markers in VMAT2 Lo PD model mice. The results demonstrate there is a significant increase in these DNA damage markers mostly in the brain regions of 18- and 23-month-old model mice, indicating oxidative stress-induced DNA lesion is an important pathologic feature of this mouse model.

Restoration of Noradrenergic Function in Parkinson's Disease Model Mice.

Dysfunction of the central noradrenergic and dopaminergic systems is the primary neurobiological characteristic of Parkinson's disease (PD). Importantly, neuronal loss in the locus coeruleus (LC) that occurs in early stages of PD may accelerate progressive loss of dopaminergic neurons. Therefore, restoring the activity and function of the deficient noradrenergic system may be an important therapeutic strategy for early PD. In the present study, the lentiviral constructions of transcription factors Phox2a/2b, Hand2 and Gata3, either alone or in combination, were microinjected into the LC region of the PD model VMAT2 Lo mice at 12 and 18 month age. Biochemical analysis showed that microinjection of lentiviral expression cassettes into the LC significantly increased mRNA levels of Phox2a, and Phox2b, which were accompanied by parallel increases of mRNA and proteins of dopamine ß-hydroxylase (DBH) and tyrosine hydroxylase (TH) in the LC. Furthermore, there was considerable enhancement of DBH protein levels in the frontal cortex and hippocampus, as well as enhanced TH protein levels in the striatum and substantia nigra. Moreover, these manipulations profoundly increased norepinephrine and dopamine concentrations in the striatum, which was followed by a remarkable improvement of the spatial memory and locomotor behavior. These results reveal that over-expression of these transcription factors in the LC improves noradrenergic and dopaminergic activities and functions in this rodent model of PD. It provides the necessary groundwork for the development of gene therapies of PD, and expands our understanding of the link between the LC-norepinephrine and dopamine systems during the progression of PD.

Clinical characteristics of a group of deaths with COVID-19 pneumonia in Wuhan, China: a retrospective case series.

BACKGROUND: With the widespread outbreak of novel coronavirus diseases 2019(COVID-19), more and more death cases were reported, however, limited data are available for the patients who died. We aimed to explore the clinical characteristics of deaths with COVID-19 pneumonia. METHODS: We abstracted and analyzed epidemiological, demographic, clinical, and laboratory data from 83 death cases with COVID-19 pneumonia in East Hospital of Wuhan University Renmin Hospital, between January 26, 2020, and February 28, 2020. RESULTS: Of the 83 deaths, none was the medical staff. The mean age was 71.8 years (SD 13.2; range, 34-97 years) and 53(63.9%) were male. The median from onset to admission was 10 days (IQR 7-14: range, 2-43 days), to death was 17 days (IQR 14-21: range, 6-54 days). Most deaths (66[80%]) had underlying comorbid diseases, the most of which was hypertension [47(57%)]. The main initial symptoms of these 83 deaths were shortness of breath(98.8%), fever(94%), and myalgia or fatigue(90.4%). Laboratory analyses showed the lymphocytopenia in 69(83%) deaths, hypoalbuminemia in 77(93%) deaths, the elevation of lactate dehydrogenase in 79(95%) deaths, procalcitonin in 69(83%) deaths and C-reactive protein in 79(95%) deaths. All 83 patients received antiviral treatment, 81(97.6%) deaths received antibiotic therapy, 54(65.1%) deaths received glucocorticoid therapy, and 20(24.1%) patients received invasive mechanical ventilation. CONCLUSION: Most of the deaths with COVID-19 pneumonia were elderly patients with underlying comorbid diseases, especially those over 70 years of age. The time of death after the onset of the disease was mostly 15-21 days. More care should be given to the elderly in further prevention and control strategies of COVID-19.

Association between fasting blood glucose and outcomes and mortality in acute ischaemic stroke patients with diabetes mellitus: a retrospective observational study in Wuhan, China.

OBJECTIVE: To evaluate the predictive value of fasting blood glucose (FBG) on unfavourable outcomes and mortality in diabetes mellitus (DM) patients after acute ischaemic stroke (AIS). STUDY DESIGN: A hospital-based observational cohort study was conducted. Clinical data, including sex, age, body mass index, vascular risk factors and systolic/diastolic blood pressure, were routinely collected. National Institutes of Health Stroke Scale score was used to assess stroke severity on admission. FBG was determined on the first day after fasting for at least 8 hours. The modified Rankin Scale was used to assess functional outcome at 90 days: 3-6, unfavourable outcome and 6, death. SETTING: Renmin Hospital of Wuhan University, Wuhan, China. PARTICIPANTS: Patients who had AIS with DM, who were consecutively admitted within 24 hours of onset from January 2018 to June 2019. RESULTS: For the 568 patients, the median age was 65 years (IQR, 55-74 years). There were 377 (66.4%) men. The median FBG values were 7.37 mmol/L (IQR, 5.99-10.10 mmol/L), and the median glycated haemoglobin (HbA1c) values were 6.6 (IQR, 5.8-8.3). Multivariable logistic and Cox regression analysis of confounding factors showed that FBG at the time of admission was an independent predictor of unfavourable outcome (OR, 1.25 (1.14-1.37); p<0.0001) and mortality (HR, 1.10 (1.03-1.15); p<0.05) at 90 days after onset. Time to death was analysed by Kaplan-Meier curves based on FBG quartiles. The risk of death in the two highest quartile groups (FBG, 7.38-10.10 mmol/L; FBG, ≥10.11 mmol/L) was significantly higher than that in the two lowest quartile groups (FBG, ≤6.00 mmol/L; FBG, 6.01-7.37 mmol/L; p<0.0001). CONCLUSIONS: Higher FBG levels are associated with unfavourable outcomes and mortality in Chinese patients who had AIS with DM. Our data contribute to the knowledge regarding the relationship between FBG and prognosis in patients with DM who had AIS.

Critical Role of Oxidatively Damaged DNA in Selective Noradrenergic Vulnerability.

An important pathology in Parkinson's disease (PD) is the earlier and more severe degeneration of noradrenergic neurons in the locus coeruleus (LC) than dopaminergic neurons in the substantia nigra. However, the basis of such selective vulnerability to insults remains obscure. Using noradrenergic and dopaminergic cell lines, as well as primary neuronal cultures from rat LC and ventral mesencephalon (VM), the present study compared oxidative DNA damage response markers after exposure of these cells to hydrogen peroxide (H2O2). The results showed that H2O2 treatment resulted in more severe cell death in noradrenergic cell lines SK-N-BE(2)-M17 and PC12 than dopaminergic MN9D cells. Furthermore, there were higher levels of oxidative DNA damage response markers in noradrenergic cells and primary neuronal cultures from the LC than dopaminergic cells and primary cultures from the VM. It included increased tail moments and tail lengths in Comet assay, and increased protein levels of phosphor-p53 and γ-H2AX after treatments with H2O2. Consistent with these measurements, exposure of SK-N-BE(2)-M17 cells to H2O2 resulted in higher levels of reactive oxygen species (ROS). Further experiments showed that exposure of SK-N-BE(2)-M17 cells to H2O2 caused an increased level of noradrenergic transporter, reduced protein levels of copper transporter (Ctr1) and 8-oxoGua DNA glycosylase, as well as amplified levels of Cav1.2 and Cav1.3 expression. Taken together, these experiments indicated that noradrenergic neuronal cells seem to be more vulnerable to oxidative damage than dopaminergic neurons, which may be related to the intrinsic characteristics of noradrenergic neuronal cells.

Norepinephrine upregulates the expression of tyrosine hydroxylase and protects dopaminegic neurons against 6-hydrodopamine toxicity.

As a classic neurotransmitter in the brain, norepinephrine (NE) also is an important modulator to other neuronal systems. Using primary cultures from rat ventral mesencephalon (VM) and dopaminergic cell line MN9D, the present study examined the neuroprotective effects of NE and its effects on the expression of tyrosine hydroxylase (TH). The results showed that NE protected both VM cultures and MN9D cells against 6-hydroxydopamine-caused apoptosis, with possible involvement of adrenal receptors. In addition, treatment with NE upregulated TH protein levels in dose- and time-dependent manner. Further experiments to investigate the potential mechanisms underlying this NE-induced upregulation of TH demonstrated a marked increase in protein levels of the brain-derived neurotrophic factor (BDNF) and the phosphorylated extracellular signal-regulated protein kinase 1 and 2 (pERK1/2) in VM cultures treated with NE. In MN9D cells, a significantly increase of TH and pERK1/2 protein levels were observed after their transfection with BDNF cDNA or exposure to BDNF peptides. Treatment of VM cultures with K252a, an antagonist of the tropomyosin-related kinase B, blocked the upregulatory effects of NE on TH, BDNF and pERK1/2. Administration of MEK1 & MEK2 inhibitors also reversed NE-induced upregulation of TH and pERK1/2. Moreover, ChIP assay showed that treatment with NE or BDNF increased H4 acetylation in the TH promoter. These results suggest that the neuroprotection and modulation of NE on dopaminergic neurons are mediated via BDNF and MAPK/ERK pathways, as well as through epigenetic histone modification, which may have implications for the improvement of therapeutic strategies for Parkinson's disease.

Elevated plasma D-dimer levels are associated with short-term poor outcome in patients with acute ischemic stroke: a prospective, observational study.

BACKGROUND: Elevated levels of plasma D-dimer increase the risk of ischemic stroke, stroke severity, and the progression of stroke status, but the association between plasma D-dimer level and functional outcome is unclear. The aim of this study is to investigate whether plasma D-dimer level is a determinant of short-term poor functional outcome in patients with acute ischemic stroke (AIS). METHODS: This prospective study included 877 Chinese patients with AIS admitted to Renmin Hospital of Wuhan University within 72 h of symptom onset. Patients were categorized by plasma D-dimer level: Quartile 1(≤0.24 mg/L), Quartile 2 (0.25-0.56 mg/L), Quartile 3 (0.57-1.78 mg/L), and Quartile 4 (> 1.78 mg/L). The medical record of each patient was reviewed, and demographic, clinical, laboratory and neuroimaging information was abstracted. Functional outcome at 90 days was assessed with the modified Rankin Scale. RESULTS: Poor outcome was present in 302 (34.4%) of the 877 patients that were included in the study (mean age, 64 years; male, 68.5%). After adjustment for potential confounding variables, higher plasma D-dimer level on admission was associated with poor outcome (adjusted odds ratio 2.257, 95% confidence interval 1.349-3.777 for Q4:Q1; P trend = 0.004). According to receiver operating characteristic (ROC) analysis, the best discriminating factor for poor outcome was a plasma D-dimer level ≥ 0.315 mg/L (area under the ROC curve 0.657; sensitivity 83.8%; specificity 41.4%). CONCLUSION: Elevated plasma D-dimer levels on admission are significantly associated with poor outcome after admission for AIS, suggesting the potential role of plasma D-dimer level as a predictive marker for short-term poor outcome in patients with AIS.

Regulating glioma stem cells by hypoxia through the Notch1 and Oct3/4 signaling pathway.

To investigate the effects of hypoxia on the features of cancer stem cells in the glioma cancer U87 cell line and underlying mechanism, stem cell markers and features in U87 were studied under the hypoxic and normoxic culture conditions by reverse transcription-quantitative polymerase chain reaction, western blot analysis, MTT, a colony formation test and flow cytometry. Compared to the normoxic group, the cluster of differentiation 133+ phenotype, clone formation rate and cell vitality were significantly elevated in U87 cells cultured in a hypoxic microenvironment. Also, the mRNA and protein expression of neurogenic locus notch homolog protein 1 (Notch1) and Oct3/4 were significantly elevated in U87 cells cultured in a hypoxic microenvironment, however, transcription factor SOX-2 expression was not significantly changed. These results indicate that hypoxia can promote the proliferation of glioma stem cells and maintain the characteristics of stem cells through the activation of Notch1 and Oct3/4 or Notch1 activation, affecting the biological characteristics of glioma cells.

The regulation of corticosteroid receptors in response to chronic social defeat.

Our previous studies demonstrated that chronic social defeat (CSD) up-regulated expression of the serotonin transporter (SERT) and norepinephrine transporter (NET) in the brain, which was mediated by corticosteroid receptors. In the present study we first analyzed the alterations of corticosteroid receptors in different brain regions after the CSD paradigm. The results showed that CSD significantly reduced glucocorticoid receptor (GR) protein levels in the CA1 and dentate gyrus of the hippocampus, as well as in central and basolateral nuclei of the amygdala, which was accompanied by the translocation of GR from cytoplasm to nuclei. CSD also markedly reduced GR mRNA levels and MR immunoreactivity in the CA1, CA3 and dentate gyrus areas of the hippocampus. Conversely, CSD pronouncedly enhanced GR mRNA and protein levels in the dorsal raphe nucleus and locus coeruleus relative to the control. As an extension of our previous studies, in situ hybridization and immunohistochemical staining demonstrated that CSD regimen caused a notable increase of SERT mRNA levels in the dorsal raphe nucleus and increased SERT immunoreactivities in CA1 and CA3 of the hippocampus, as well as those in the basolateral nuclei of the amygdala. Likewise, CSD regimen resulted in an evident enhancement of NET immunoreactivity in the CA1 of the hippocampus and in the basolateral nuclei of the amygdala. Our current findings suggest that GR expressional alterations in response to CSD are complex and brain region-specific, which may correspond to their different functions in these regions.

Reduction of hippocampal N-acetyl aspartate level in aged APP(Swe)/PS1(dE9) transgenic mice is associated with degeneration of CA3 pyramidal neurons.

Age-related metabolic changes in the hippocampus of APP(Swe)/PS1(dE9) mice were measured with long echo-time in vivo (1)H-magnetic resonance spectroscopy ((1)H-MRS). Thioflavine S staining and Nissl staining were used to characterize deposition of Aß aggregates and neuronal degeneration in the transgenic animals, respectively. The results showed that the APP(Swe)/PS1(dE9) mice had significantly decreased hippocampal N-acetyl aspartate (NAA)/total creatine (tCr) level at 16 months of age, which was associated with degeneration of and intracellular deposition of thioflavine S-positive materials in hippocampal CA3 pyramidal neurons. The results of this study provide direct evidence showing association among Aß pathology (intracellular deposition of thioflavine S-positive materials), neuronal degeneration, and metabolic changes observable with in vivo (1)H-MRS in the hippocampus of APP(Swe)/PS1(dE9) mice.

Role of leukaemia inhibitory factor in the induction of pluripotent stem cells in mice.

iPS (induced pluripotent stem) cells can be induced from somatic cells in mice by genetic manipulation. Most previously established mouse iPS cell lines have been derived using feeder layers supplemented with exogenous LIF (leukaemia inhibitory factor). Although a feeder-free induction system has been developed in recent studies, LIF is still required for reprogramming, but its role in the generation of mouse iPS cells has remained elusive. In this study, we investigated its contribution to the induction of pluripotency. Our results showed that LIF activates AP (alkaline phosphatase) through a c-Myc-dependent mechanism. Moreover, it acts as a protective factor during the transition from AP-positive colonies to Oct3/4-positive cells. These findings illustrate a mechanism by which LIF may integrate signalling into reprogramming.

Effect of VEGF on neural differentiation of human embryonic stem cells in vitro.

The effects of vascular endothelial growth factor (VEGF) on neural differentiation of human embryonic stem cells (hESCs) in vitro and the possible mechanism were observed. The hESCs lines, TJMU1 and TJMU2, were established and stored by our laboratory. hESCs differentiated into neuronal cells through embryonic body formation. In this induction process, hESCs were divided into three groups: group A, routine induction; group B, routine induction+10 ng/mL VEGF; group C, routine induction+10 ng/mL VEGF+10 ng/mL VEGFR2/Fc. OCT4, Nestin and GFAP in each group were detected by RT-PCR, and the cells expressing Nestin and GFAP were counted by immunofluorescence. The percentage of Nestin positive cells in group B was significantly higher than in groups A and C, while the percentage of GFAP positive cells in group B was significantly lower than in groups A and C (P<0.01). There was no significant difference between groups A and C (P>0.05). It was concluded that VEGF, via VEGFR2, stimulated the neural differentiation of hESCs in vitro.

Effect of VEGF on Neural Differentiation of Human Embryonic Stem Cells in vitro / 华中科技大学学报（医学）（英德文版）

The effects of vascular endothelial growth factor (VEGF) on neural differentiation of human embryonic stem cells (hESCs) in vitro and the possible mechanism were observed. The hESCs lines,TJMU1 and TJMU2, were established and stored by our laboratory, hESCs differentiated into neuronal cells through embryonic body formation. In this induction process, hESCs were divided into three groups: group A, routine induction; group B, routine induction+10 ng/mL VEGF; group C, routine in-duction+10 ng/mL VEGF+10 ng/mL VEGFR2/Fc. OCT4, Nestin and GFAP in each group were de-tected by RT-PCR, and the cells expressing Nestin and GFAP were counted by immunofluorescence.The percentage of Nestin positive cells in group B was significantly higher than in groups A and C,while the percentage of GFAP positive cells in group B was significantly lower than in groups A and C (P<0.01). There was no significant difference between groups A and C (P>0.05). It was concluded that VEGF, via VEGFR2, stimulated the neural differentiation of hESCs in vitro.

[Study on distribution of neural stem cells in the brain of Alzheimer disease transgenic mice through caudal vein transplantation].

OBJECTIVE: To observe whether neural stem cells (NSCs) can successfully permeate into the brain through the blood-brain barrier (BBB) of Alzheimer disease (AD) transgenic mice and explore the methods of distribution and migration. METHODS: NSCs were isolated from 12-day-old fetal mice, cultured, labeled with enhanced green fluorescent protein (eGFP) and then transplanted into 10 AD transgenic mice and normal mice as controls through caudal vein. The mice were killed 48 h, 1 w, 2 w, and 4 w after transplantation respectively. The brains of the mice were made into continual frozen sections, the distribution and migration of the eGFP-labeled NSCs were studied under fluorescence microscope. RESULTS: At different time points after transplantation the eGFP-labeled NSCs were diffusely distributed in the brain: distributed around the blood vessels in the first 48 h, and then migrated gradually towards the hippocampus and cortex until 4 weeks later. There were no obvious abnormal complications occurring after transplantation. CONCLUSION: NSCs can successfully permeate into the brain through the BBB of AD transgenic mice, and migrate into the brain parenchyma gradually.