scholarly journals Induction of Parkinson Disease-Related Proteins in Motor Neurons after Transient Spinal Cord Ischemia in Rabbits

2009 ◽  
Vol 29 (4) ◽  
pp. 752-758 ◽  
Author(s):  
Masahiro Sakurai ◽  
Takae Kawamura ◽  
Hidekazu Nishimura ◽  
Hiroyoshi Suzuki ◽  
Fumiaki Tezuka ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Western Blot ◽  
Motor Neurons ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Early Stage ◽  
Spinal Cord Ischemia ◽  
Balloon Catheter ◽  
Cord Injury ◽  
Double Label

The mechanism of spinal cord injury has been thought to be related to the vulnerability of spinal motor neuron cells against ischemia. However, the mechanisms of such vulnerability are not fully understood. We investigated a possible mechanism of neuronal death by immunohistochemical analysis for DJ-1, PINK1, and α-Synuclein. We used a 15-min rabbit spinal cord ischemia model, with use of a balloon catheter. Western blot analysis for DJ-1, PINK1, and α-Synuclein; temporal profiles of DJ-1, PINK1, and α-Synuclein immunoreactivity; and double-label fluorescence immunocytochemical studies were performed. Western blot analysis revealed scarce immunoreactivity for DJ-1, PINK1, and α-Synuclein in the sham-operated spinal cords. However, they became apparent at 8 h after transient ischemia, which returned to the baseline level at 1 day. Double-label fluorescence immunocytochemical study revealed that both DJ-1 and PINK1, and DJ-1 and α-Synuclein were positive at 8 h of reperfusion in the same motor neurons, which eventually die. The induction of DJ-1 and PINK1 proteins in the motor neurons at the early stage of reperfusion may indicate oxidative stress, and the induction of α-Synuclein may be implicated in the programmed cell death change after transient spinal cord ischemia.

Arsenic Trioxide Affects Early Stage Angiogenesis through Inhibition of CD14 Monocyte Transdifferentiation into Endothelial Cells Induced by Pleiotrophin and mCSF.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1267-1267
Author(s):  
Haiming Chen ◽  
Mingjie Li ◽  
Richard A. Campbell ◽  
Melinda S. Gordon ◽  
Dror Shalitin ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Western Blot ◽  
Arsenic Trioxide ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Early Stage ◽  
Vascular Endothelial ◽  
Cam Assay ◽  
Rt Pcr ◽  
Vessel Formation

Abstract We have discovered a novel mechanism leading to blood vessel formation involving transdifferentiation of monocytes into endothelial cells by tumor cell production of pleiotrophin (PTN), a protein highly produced by myeloma (H. Chen et al, Blood, 2005; Yeh et al BJH, 2006). Arsenic trioxide (ATO) induces apoptosis of cancer cells directly through a number of mechanisms, and this drug has also been shown to inhibit angiogenesis. However, it remains unknown whether ATO affects the earliest stages of angiogenesis and vasculogenesis important in tumor development. We purified human monocytes (CD14+) and cultured these cells on collagen I-coated dishes. mCSF was added to the cells after 1 hour of culture. PTN was added twice to the culture, once after 24 hours and again after 5 days with or without ATO or bortezomib. FLK-1 expression (VEGFR-2) showed that the cells incubated on collagen I without drugs formed tube-like structures in the presence of PTN and mCSF. However, the tube-like structures disappeared after adding either the IC50 (5x10−6M) dose or low (5x10−7M) dose of ATO. FLK-1 staining remains in the tube-like structures with low doses (3x10−12M) of bortezomib. In order to examine whether ATO or bortezomib affects endothelial gene expression when monocytes are induced to transdifferentiate in the presence of these cytokines, we also examined expression using RT-PCR on endothelial cell genes (vascular endothelial growth factor receptor-2 (Flk-1), Tie-2 and von Willebrand factor (vWF)) and Western blot analysis for protein expression. The results of both RT-PCR and Western blot analysis showed that the expression of endothelial markers was blocked at both the higher (5x10−6M) and lower (5x10−7M) doses of ATO. In contrast, the expression of endothelial markers was not reduced by adding low dose bortezomib (3x10−12M). We further examined the effects of ATO and bortezomib on early stage angiogenesis in vivo using the chorioallantoic membrane (CAM) assay. Fertilized chick eggs were incubated horizontally at 38°C in a humidified incubator, windowed by day 3 of incubation and processed by day 8. The tested micro-sponge with ATO (5x10−6M) or bortezomib (3x10−11M) or control reagents was implanted on the CAM. The eggs were sealed with adhesive tape and returned to the incubator for 48 hours. The assay scored positive when two independent observers reported a significant reduction of vessels in the treated area. The results of the CAM assay showed that compared to saline, ATO significantly reduced new macroscopic and microscopic vessel formation. In contrast, bortezomib did not affect angiogenesis in the CAM assay. These experiments define a previously unrecognized novel mechanism by which ATO may have anti-angiogenetic effects in cancer patients-preventing the transdifferentiation of monocytes into endothelial cells by PTN. They also suggest ATO as a potential new specific agent to inhibit angiogenesis resulting from transdifferentiation of monocytes into vascular endothelial cells driven by pleiotrophin and mCSF. These results suggest a novel way by which anti-cancer agents may impact angiogenesis.

Flow Cytometric Detection of ZAP-70 in Chronic Lymphocytic Leukemia: Correlation With Immunocytochemistry and Western Blot Analysis

2007 ◽  
Vol 131 (1) ◽  
pp. 50-56
Author(s):  
Graham W. Slack ◽  
Juanita Wizniak ◽  
Laith Dabbagh ◽  
Xinzhe Shi ◽  
Pascal Gelebart ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Early Stage ◽  
Lymphocytic Leukemia ◽  
Early Stage Disease ◽  
Stage Disease ◽  
Specimen Processing

Abstract Context.—Expression of ZAP-70 in chronic lymphocytic leukemia (CLL) predicts worse clinical outcome in patients with early-stage disease. It has become important to include ZAP-70 in the immunophenotyping panel used to diagnose CLL, commonly performed by flow cytometry (FC). Nevertheless, the methodology used to detect ZAP-70 by FC has not been extensively evaluated. Objective.—To describe our FC method for detecting ZAP-70 in CLL and assess whether this assay is useful in estimating the ZAP-70 protein level in CLL cells. Design.—ZAP-70 expression was assessed by FC in 45 consecutive newly diagnosed CLL patients, and the results were correlated with those of immunocytochemistry and Western blot analysis. Results.—With >25% ZAP-70–positive B cells as the cutoff, the FC results had a perfect concordance with those of immunocytochemistry (39/39, 100%) and Western blot analysis (7/7, 100%). The use of autofluorescence controls was found to be superior to other alternatives. Overall, 19 (42%) of 45 cases were ZAP-70 positive in our series. Since only 7 cases (16%) had >20% to 30% ZAP-70–positive B cells, the cutoff of >25% readily separated CLL into positive and negative groups in most cases. ZAP-70 positivity was significantly associated with atypical morphology but not other laboratory parameters evaluated. Conclusions.—With proper specimen processing and the use of directly fluorescence-conjugated anti–ZAP-70 antibody, one can readily incorporate ZAP-70 into the routine FC study panel for CLL. Our data suggest that FC is a rapid and useful method to estimate the ZAP-70 protein expression level in CLL.

scholarly journals mRNA and miRNA expression profile reveals the role of miR-31 overexpression in neural stem cell

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Pengfei Li ◽  
Yuantao Gao ◽  
Xiao Li ◽  
Feng Tian ◽  
Fei Wang ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Motor Neurons ◽  
Early Stage ◽  
Expression Profiles ◽  
Mirna Expression Profile ◽  
Control Group ◽  
Cord Injury ◽  
Candidate Target ◽  
Mirnas Expression

Abstract A detailed understanding of the character and differentiation mechanism of neural stem cells (NSCs) will help us to effectively utilize their transplantation to treat spinal cord injury. In previous studies, we found that compared with motor neurons (MNs), miR-31 was significantly high-expressed in NSCs and might play an important role in the proliferation of NSCs and the differentiation into MNs. To better understand the role of miR-31, we characterized the mRNA and miRNAs expression profiles in the early stage of spinal cord-derived NSCs after miR-31 overexpression. There were 35 mRNAs and 190 miRNAs differentially expressed between the miR-31 overexpression group and the control group. Compared with the control group, both the up-regulated mRNAs and miRNAs were associated with the stemness maintenance of NSCs and inhibited their differentiation, especially to MNs, whereas the down-regulated had the opposite effect. Further analysis of the inhibition of miR-31 in NSCs showed that interfering with miR-31 could increase the expression of MNs-related genes and produce MNs-like cells. All these indicated that miR-31 is a stemness maintenance gene of NSCs and has a negative regulatory role in the differentiation of NSCs into MNs. This study deepens our understanding of the role of miR-31 in NSCs, provides an effective candidate target for effectively inducing the differentiation of NSCs into MNs, and lays a foundation for the effective application of NSCs in clinic.

scholarly journals Effect of Chinese herbal compound GAPT on the early brain glucose metabolism of APP/PS1 transgenic mice

2019 ◽  
Vol 33 ◽  
pp. 205873841984148 ◽  
Author(s):  
Lulu Mana ◽  
Huili Feng ◽  
Yunfang Dong ◽  
Yahan Wang ◽  
Jing Shi ◽  
...  
Keyword(s):  
Glucose Metabolism ◽  
Transgenic Mice ◽  
Western Blot ◽  
Pet Imaging ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Early Stage ◽  
Model Group ◽  
Brain Glucose ◽  
Brain Glucose Metabolism

A number of studies have shown that early-stage Alzheimer’s disease (AD) is associated with abnormal brain glucose metabolism before cognitive decline, which may be the key pathological change of asymptomatic AD. The pathogenesis of AD in traditional Chinese medicine is kidney deficiency and turbid phlegm. Based on this, GAPT (a mixture of herbal extracts) was made to invigorate kidney Yang and eliminate phlegm. Previous studies have shown that GAPT can improve and delay the memory decline, but the specific therapeutic target of AD in an early stage has not been studied. The aim of this study was to investigate the effect of GAPT on glucose metabolism in the early stage of AD. Eighty-eight 3-month-old male APP/PS1 transgenic mice were randomly divided into model group; donepezil group; and low, middle and high GAPT dosage groups. Twelve 3-month-old C57BL/6J mice were used as a control group. The Morris water maze test and the Step-Down Passive-Avoidance test were used to evaluate learning and memory ability. Cerebral extraction and the accumulation of glucose were scanned with a micro-positron-emission tomography (PET) imaging system. Immunohistochemistry, western blot analysis and polymerase chain reaction (PCR) were used to detect the expression of the PI3K/AKT-mTOR signalling pathway–related proteins and messenger RNAs (mRNAs) in hippocampus of APP/PS1 transgenic mice after 3 months of drug administration. GAPT can shorten the escape latency and error numbers compared to the model group. In micro-PET imaging analysis, GAPT can increase the glucose uptake average rate in the frontal lobe, temporal lobe, parietal lobe and hippocampus. The immunohistochemistry, western blot analysis and PCR results indicated that GAPT can increase the expression of PI3K, AKT, GLUT1 and GLUT3 in the hippocampus of APP/PS1 transgenic mice. In summary, GAPT can improve brain glucose metabolism damage in APP/PS1 transgenic mice, mainly by increasing brain glucose uptake, increasing glucose transport and improving the insulin signalling pathway.

scholarly journals Profound Hypotension before Aortic Clamping Can Exacerbate Spinal Cord Ischemic Injury after Aortic Surgery in Rats

2020 ◽  
Vol 9 (11) ◽  
pp. 3395
Author(s):  
Chang-Hoon Koo ◽  
Jung-Hee Ryu ◽  
Jin-Young Hwang ◽  
Jin-Hee Kim ◽  
Hyun-Jung Shin ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Motor Neurons ◽  
Histopathological Examination ◽  
Aortic Surgery ◽  
Spinal Cord Ischemia ◽  
Motor Deficit ◽  
Control Group ◽  
Aortic Clamping ◽  
Cord Injury ◽  
Profound Hypotension

Spinal cord ischemia is one of the most serious complications of aortic repair in patients with acute aortic syndrome. However, the effect of hypotension before aortic clamping on spinal cord injury has not been documented. A total of 48 male Sprague-Dawley rats were randomly divided into four groups: the sham group; control group (mean arterial pressure (MAP) < 90% of baseline value before aortic clamping); mild hypotension group (MAP < 80%); and profound hypotension group (MAP < 60%). Spinal cord ischemia was induced using a balloon-tipped catheter placed in the descending thoracic aorta. Neurological function of the hind limbs was evaluated for seven days after reperfusion and recorded using a motor deficit index (MDI). The spinal cord was then harvested for histopathological examination and evaluation of oxidative stress and inflammation. The profound hypotension group demonstrated a significantly higher MDI 48 h post-reperfusion and lower number of normal motor neurons than the other groups (p < 0.001). The levels of tissue malondialdehyde and tumor necrosis factor-α (TNF-α) were also significantly increased in the profound hypotension group compared with other groups. Profound hypotension before aortic clamping can aggravate neurologic outcomes after aortic surgery by exacerbating neurologic injury and reducing the number of normal motor neurons.

Expression of basic fibroblast growth factor in the nervous system of early avian embryos

Development ◽  
1990 ◽  
Vol 109 (1) ◽  
pp. 203-215 ◽  
Author(s):  
C. Kalcheim ◽  
G. Neufeld
Keyword(s):  
Spinal Cord ◽  
Nervous System ◽  
Growth Factor ◽  
Western Blot ◽  
Neural Crest ◽  
Fibroblast Growth Factor ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Fibroblast Growth ◽  
Specific Staining

Basic fibroblast growth factor (bFGF) promotes the survival of a subpopulation of non-neuronal cells developing from trunk neural crest. It was therefore important to determine whether this factor is present in the nervous system at early developmental stages. Immunocytochemistry using specific polyclonal and monoclonal antibodies was combined with three highly sensitive assays: bFGF-induced proliferation of bovine adrenal cortex-derived capillary endothelial cells (ACE), a radioimmunoassay for bFGF (RIA) and Western blot analysis. bFGF immunoreactivity was localized to the cytoplasm of neuroepithelial cells derived from embryonic day 2 (E2) quail neural tubes and cultured for one day in a chemically defined medium. Specific staining was observed in young sensory neurons in cultures of neural crest clusters as well as in a subpopulation of non-neuronal cells. In cultured E7 dorsal root ganglia, immunostaining was confined to neuronal cell bodies and fibers. In situ, staining of spinal cord and ganglionic neurons appeared on E6 and increased in intensity towards E10. Various mesoderm-derived structures such as the limb buds, the mesenchyme dorsal to the neural tube, the vertebral muscles and cartilage showed specific staining patterns in addition to neural tissue. In agreement with the results of immunocytochemical studies, 1.4ng bFGF per mg protein was detected in spinal cord extracts by RIA as early as E3, its concentration increased to 8.0 ng mg-1 on E5 and then to a maximum of 18.0 ng mg-1 protein on E10, this was followed by a subsequent decrease in concentration in older embryos. On the other hand, high levels of bFGF were present in vertebral tissues from E10 onwards. Extracts of immunopositive tissues were subjected to heparin-Sepharose affinity chromatography and eluted in a stepwise salt gradient. Fractions that eluted from the columns at 2 M NaCl contained a bFGF-like protein as revealed by their ability to stimulate the proliferation of ACE cells and by Western blot analysis. These data demonstrate that bFGF is expressed during early nervous system development in both central and peripheral neurons.

scholarly journals Lycium Barbarum Polysaccharides Alleviates Oxidative Damage Induced by H2O2 Through Down-Regulating MicroRNA-194 in PC-12 and SH-SY5Y Cells

2018 ◽  
Vol 50 (2) ◽  
pp. 460-472 ◽  
Author(s):  
Tong Niu ◽  
Liuzhong Jin ◽  
Shizhen Niu ◽  
Cunqi Gong ◽  
Hui Wang
Keyword(s):  
Oxidative Stress ◽  
Flow Cytometry ◽  
Western Blot ◽  
Cell Viability ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Akt Pathway ◽  
Lycium Barbarum ◽  
Cord Injury ◽  
Lycium Barbarum Polysaccharides

Background/Aims: Currently, scientists attempt to improve outcome of spinal cord injury (SCI) via reducing secondary injury during SCI. Oxidative stress is critical for pathophysiology of secondary damage, thus we mainly focused on the anti-oxidant effects of Lycium barbarum polysaccharides (LBPs) on PC-12 and SH-SY5Y cells as well as the underlying mechanisms. Methods: Oxidative stress was induced by H2O2 stimulation. Effects of LBPs on cell viability, apoptosis, and expression of proteins associated with apoptosis and autophagy in H2O2-induced cells were assessed by CCK-8 assay, flow cytometry assay and Western blot analysis, respectively. Then, expression of miR-194 was determined by qRT-PCR. Expression of miR-194 was dysregulated, and whether LBPs affected H2O2-treated cells through modulating miR-194 was verified. The expression of key kinases in the PI3K/AKT pathway and the intracellular levels of ROS and NO were testified by Western blot analysis and flow cytometry with fluorescent probes. Results: H2O2-induced decrease of cell viability and increases of apoptosis and autophagy in PC-12 cells were mitigated by LBPs treatment. Next, we found that miR-194 expression was both down-regulated by LBPs treatment in PC-12 and SH-SY5Y cells. More experiments consolidated that influence of LBPs on H2O2-treated cells was reversed by miR-194 overexpression while was augmented by miR-194 inhibition. LBPs elevated the phosphorylated levels of PI3K and AKT and reduced levels of ROS and NO through miR-194. Conclusion: LBPs alleviated H2O2-induced decrease of cell viability, and increase of apoptosis and autophagy through down-regulating miR-194. Moreover, LBPs activated the PI3K/AKT pathway and reduced oxidative stress through miR-194.

Effects of HBO with reduction of iNOS and HIF -1α in motor neurons after transient spinal cord ischemia in rabbits

2005 ◽  
Vol 25 (1_suppl) ◽  
pp. S452-S452
Author(s):  
Noritaka Murakami ◽  
Masahiro Sakurai ◽  
Takashi Horinouchi ◽  
Jun Ito ◽  
Shin Kurosawa ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Motor Neurons ◽  
Spinal Cord Ischemia
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