scholarly journals CORM-2 can Attenuate Bleeding-mediated Inflammation by Increasing Phagocytic Capacity of Cerebral Microglial Cells in Neonatal Rat in Vitro

2021 ◽  
Keyword(s):  
Neonatal Rat ◽  
Microglial Cells ◽  
Phagocytic Capacity

Abstract The authors have requested that this preprint be withdrawn due to a need to make corrections.

scholarly journals CORM-2 can Attenuate Bleeding-mediated Inflammation by Increasing Phagocytic Capacity of Cerebral Microglial Cells in Neonatal Rat in Vitro

2021 ◽  
Author(s):  
Zhiying Chen ◽  
Huiyan Zhang ◽  
Jun Zhou ◽  
Xiaoqin Wu ◽  
Moxin Wu ◽  
...  
Keyword(s):  
Treated Group ◽  
Neonatal Rat ◽  
Neonatal Rats ◽  
Phagocytic Capacity ◽  
Inflammatory Reactions ◽  
Microglial Phagocytosis ◽  
Protein Expressions ◽  
Cell Groups ◽  
Cell Supernatant

Abstract Objective: This study aimed to explore the mechanism of CORM-2 on attenuating bleeding-related inflammation. Methods: Microglia were isolated from the neonatal rats (1-2days old) and identified by the CD11b/c anti-body, and some microglia were co-cultured with RBCs marked with PKH26 fluorescent dye, and then treated with CORM-2. That is, the microglia cells were divided into the microglia, microglia+ PKH26+RBCs and microglia + PKH26+ RBCs+CORM-2 cell-groups. Microglial phagocytosis to RBCs PKH26+ was observed under an inverted fluorescence microscope; moreover, the fluorescence intensity of microglia that phagocytized PKH26+RBCs was detected through immunofluorescence. HO-1, NF-κB p65, and IL-1β expressions were detected using RT-qPCR, western blotting, and immunofluorescence, respectively. The levels of carbon monoxide hemoglobin (HbCO) in the cell supernatant in each group were detected with ELISA.Results. After 1- day of co-culturing, the number of residual PKH26+RBCs in the Microglia+ PKH26+RBCs+CORM-2 group decreased remarkably than that in the Microglia+ PKH26+RBCs groups (18 × 106 vs. 14 × 106, p=0.02), which revealed that microglia phagocytosis was stronger in CORM-2 treated group. More over, compared with microglia + PKH26+RBCs group, the microglia+ PKH26+RBCs +CORM-2 group showed higher levels of HO-1 mRNA and protein expressions at the 3rd day and the 5th day after co-culturing. Further more, CORM-2 significantly inhibited the expressions of mRNA and proteins of NF‐κB p65 and IL-1 after 3 days of co-culturing, meanwhile, CORM-2 did not increase the level of HbCO in the cell supernatant.Conclusions CORM-2 can inhibit inflammatory reactions in bleeding setting in vitro by promoting microglial phagocytosis to RBCs and decrease IL-1β and NF-κB; the mechanism may involve HO-1/CO system.

scholarly journals Microglia Stimulation by Protein Extract of Injured Rat Spinal Cord. A Novel In vitro Model for Studying Activated Microglia

2021 ◽  
Vol 14 ◽  
Author(s):  
Joaquim Hernández ◽  
Isaac Francos-Quijorna ◽  
Elena Redondo-Castro ◽  
Rubén López-Vales ◽  
Xavier Navarro
Keyword(s):  
Spinal Cord ◽  
Inflammatory Cytokines ◽  
Microglial Activation ◽  
Microglial Cells ◽  
Phagocytic Capacity ◽  
Contusion Injury ◽  
Spinal Cord Contusion ◽  
Inflammatory Profile ◽  
Set Up

Research on microglia has established the differentiation between the so-called M1 and M2 phenotypes. However, new frameworks have been proposed attempting to discern between meaningful microglia profiles. We have set up an in vitro microglial activation model by adding an injured spinal cord (SCI) lysate to microglial cultures, obtained from postnatal rats, in order to mimic the environment of the spinal cord after injury. We found that under the presence of the SCI lysate microglial cells changed their phenotype, developing less ramified but longer processes, and proliferated. The SCI lysate also led to upregulation of pro-inflammatory cytokines, such as IL-1β, IL-6, and TNF-α, downregulation of the anti-inflammatory cytokines IL-10 and IL-4, and a biphasic profile of iNOS. In addition, a latex beads phagocytosis assay revealed the SCI lysate stimulated the phagocytic capacity of microglia. Flow cytometry analysis indicated that microglial cells showed a pro-inflammatory profile in the presence of SCI lysate. Finally, characterization of the microglial activation in the spinal cord on day 7 after contusion injury, we showed that these cells have a pro-inflammatory phenotype. Overall, these results indicate that the use of SCI lysates could be a useful tool to skew microglia towards a closer phenotype to that observed after the spinal cord contusion injury than the use of LPS or IFNγ.

Cytochemical Evidence for Presynatic β-Adrenergic Regulation of Secretion in the Developing Rat Submandibular Gland

1977 ◽  
Vol 35 ◽  
pp. 430-431
Author(s):  
L.S. Cutler
Keyword(s):  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
Submandibular Gland ◽  
Neonatal Rat ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Parotid Glands ◽  
Adrenergic Agonist ◽  
Rat Submandibular Gland

Many studies previously have shown that the B-adrenergic agonist isoproterenol and the a-adrenergic agonist norepinephrine will stimulate secretion by the adult rat submandibular (SMG) and parotid glands. Recent data from several laboratories indicates that adrenergic agonists bind to specific receptors on the secretory cell surface and stimulate membrane associated adenylate cyclase activity which generates cyclic AMP. The production of cyclic AMP apparently initiates a cascade of events which culminates in exocytosis. During recent studies in our laboratory it was observed that the adenylate cyclase activity in plasma membrane fractions derived from the prenatal and early neonatal rat submandibular gland was retractile to stimulation by isoproterenol but was stimulated by norepinephrine. In addition, in vitro secretion studies indicated that these prenatal and neonatal glands would not secrete peroxidase in response to isoproterenol but would secrete in response to norepinephrine. In contrast to these in vitro observations, it has been shown that the injection of isoproterenol into the living newborn rat results in secretion of peroxidase by the SMG (1).

The in vitro neonatal rat spinal cord preparation: a new insight into mammalian locomotor mechanisms

2004 ◽  
Vol 190 (5) ◽  
pp. 343-357 ◽  
Author(s):  
F. Clarac ◽  
E. Pearlstein ◽  
J. F. Pflieger ◽  
L. Vinay
Keyword(s):  
Spinal Cord ◽  
Neonatal Rat ◽  
Rat Spinal Cord ◽  
Insight Into

scholarly journals Effect of memantine, an anti-Alzheimer’s drug, on rodent microglial cells in vitro

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Toru Murakawa-Hirachi ◽  
Yoshito Mizoguchi ◽  
Masahiro Ohgidani ◽  
Yoshinori Haraguchi ◽  
Akira Monji
Keyword(s):  
Neuronal Death ◽  
Phagocytic Activity ◽  
Microglial Cell ◽  
Cell Function ◽  
Microglial Cells ◽  
Phagocytosis Assay ◽  
Intracellular Ca ◽  
Β Amyloid ◽  
The Brain

AbstractThe pathophysiology of Alzheimer’s disease (AD) is related to neuroinflammatory responses mediated by microglia. Memantine, an antagonist of N-methyl-d-aspartate (NMDA) receptors used as an anti-Alzheimer’s drug, protects from neuronal death accompanied by suppression of proliferation and activation of microglial cells in animal models of AD. However, it remains to be tested whether memantine can directly affect microglial cell function. In this study, we examined whether pretreatment with memantine affects intracellular NO and Ca2+ mobilization using DAF-2 and Fura-2 imaging, respectively, and tested the effects of memantine on phagocytic activity by human β-Amyloid (1–42) phagocytosis assay in rodent microglial cells. Pretreatment with memantine did not affect production of NO or intracellular Ca2+ elevation induced by TNF in rodent microglial cells. Pretreatment with memantine also did not affect the mRNA expression of pro-inflammatory (TNF, IL-1β, IL-6 and CD45) or anti-inflammatory (IL-10, TGF-β and arginase) phenotypes in rodent microglial cells. In addition, pretreatment with memantine did not affect the amount of human β-Amyloid (1–42) phagocytosed by rodent microglial cells. Moreover, we observed that pretreatment with memantine did not affect 11 major proteins, which mainly function in the phagocytosis and degradation of β-Amyloid (1–42), including TREM2, DAP12 and neprilysin in rodent microglial cells. To the best of our knowledge, this is the first report to suggest that memantine does not directly modulate intracellular NO and Ca2+ mobilization or phagocytic activity in rodent microglial cells. Considering the neuroinflammation hypothesis of AD, the results might be important to understand the effect of memantine in the brain.

scholarly journals Conditioned medium-preconditioned EPCs enhanced the ability in oligovascular repair in cerebral ischemia neonatal rats

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Ning Zhou ◽  
Lei Wang ◽  
Ping Fu ◽  
Zihao Cui ◽  
Yuhang Ge ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Western Blot ◽  
Conditioned Medium ◽  
Cognitive Outcome ◽  
Adult Brain ◽  
Neonatal Rat ◽  
Vascular Density ◽  
Cxcr4 Axis

Abstract Background Oligovascular niche mediates interactions between cerebral endothelial cells and oligodendrocyte precursor cells (OPCs). Disruption of OPC-endothelium trophic coupling may aggravate the progress of cerebral white matter injury (WMI) because endothelial cells could not provide sufficient support under diseased conditions. Endothelial progenitor cells (EPCs) have been reported to ameliorate WMI in the adult brain by boosting oligovascular remodeling. It is necessary to clarify the role of the conditioned medium from hypoxic endothelial cells preconditioned EPCs (EC-pEPCs) in WMI since EPCs usually were recruited and play important roles under blood-brain barrier disruption. Here, we investigated the effects of EC-pEPCs on oligovascular remodeling in a neonatal rat model of WMI. Methods In vitro, OPC apoptosis induced by the conditioned medium from oxygen-glucose deprivation-injured brain microvascular endothelial cells (OGD-EC-CM) was analyzed by TUNEL and FACS. The effects of EPCs on EC damage and the expression of cytomokine C-X-C motif ligand 12 (CXCL12) were examined by western blot and FACS. The effect of the CM from EC-pEPCs against OPC apoptosis was also verified by western blot and silencing RNA. In vivo, P3 rat pups were subjected to right common carotid artery ligation and hypoxia and treated with EPCs or EC-pEPCs at P7, and then angiogenesis and myelination together with cognitive outcome were evaluated at the 6th week. Results In vitro, EPCs enhanced endothelial function and decreased OPC apoptosis. Meanwhile, it was confirmed that OGD-EC-CM induced an increase of CXCL12 in EPCs, and CXCL12-CXCR4 axis is a key signaling since CXCR4 knockdown alleviated the anti-apoptosis effect of EPCs on OPCs. In vivo, the number of EPCs and CXCL12 protein level markedly increased in the WMI rats. Compared to the EPCs, EC-pEPCs significantly decreased OPC apoptosis, increased vascular density and myelination in the corpus callosum, and improved learning and memory deficits in the neonatal rat WMI model. Conclusions EC-pEPCs more effectively promote oligovascular remodeling and myelination via CXCL12-CXCR4 axis in the neonatal rat WMI model.

“Trapped reentry” as a dormant source of acute focal arrhythmia and fractionated atrial electrograms under sinus rhythm

2020 ◽  
Vol 41 (Supplement_2) ◽  
Author(s):  
T De Coster ◽  
A.S Teplenin ◽  
I Feola ◽  
T.J Van Brakel ◽  
A.A.F De Vries ◽  
...  
Keyword(s):  
Sinus Rhythm ◽  
Acute Onset ◽  
Neonatal Rat ◽  
Funding Source ◽  
Geometrical Properties ◽  
Conduction Pathway ◽  
Atrial Electrograms ◽  
Public Grant ◽  
Source Sink

Abstract Background Diseased atria are characterised by functional and structural heterogeneities (e.g. dense fibrotic regions), which add to abnormal impulse generation and propagation, like ectopy and block. These heterogeneities are thought to play a role in the origin of complex fractionated atrial electrograms (CFAEs) under sinus rhythm (SR) in atrial fibrillation (AF) patients, but also in the onset and perpetuation (e.g. reentry) of this disorder. The underlying mechanisms, however, remain incompletely understood. Purpose To test the hypothesis that dense local fibrotic regions could create an electrically isolated conduction pathway in which reentry can be established via ectopy and block to become “trapped” (giving rise to CFAEs under SR), only to be “released” under dynamic changes at a connecting isthmus (causing acute focal arrhythmia (FA)). Methods The geometrical properties of such an electrically isolated pathway, under which reentry could be trapped and released, were explored in vitro using optogenetics by creating conduction blocks of any shape by means of light-gated depolarizing ion channels (CatCh) and patterned illumination. Insight from these studies was used for complementary computational investigation in virtual human atria to assess clinical translation and to provide deeper mechanistic insight. Results Optical mapping studies, in monolayers of CatCh-activated neonatal rat atrial cardiomyocytes, revealed that reentry could indeed be established and trapped by creating an electrically isolated pathway with a connecting isthmus causing source-sink mismatch. This proves that a tachyarrhythmia can exist locally with SR prevailing in the bulk of the monolayer. Next, it was confirmed under which conditions reentry could escape this pathway by widening of the isthmus (i.e. overcoming the source-sink mismatch), thereby converting this local dormant arrhythmic source into an active driver with global impact (i.e. acute monolayer-wide FA). This novel phenomenon was shown in circuits <0.7cm2, adding to their probability to exist in human atria. Computational 3D studies revealed that the conditions for “trapped reentry” and its release can indeed be realized in human atria. Unipolar epicardial pseudo-electrograms derived from these simulations showed CFAEs at the site of “trapped reentry” in coexistence with normal electrograms showing SR in the bulk of the atria. Upon release of the reentry through reduction of gap junctional coupling, acute FA occurred, affecting the complete atria as evidenced by wave front and electrogram visualization. Conclusion This study reveals that “trapped reentry”, a previously undesignated phenomenon, can explain the origin of two designated ones: the observation of CFAEs under SR and acute onset of FA. Further exploration of the concept of “trapped reentry” may not only expand our understanding of AF initiation and perpetuation, but also termination, including ablation strategies by site-directed targeting. Funding Acknowledgement Type of funding source: Public grant(s) – EU funding. Main funding source(s): This study was funded by the European Research Council (Starting grant 716509) to D.A. Pijnappels

scholarly journals Knockdown of long non-coding RNA LEF1-AS1 attenuates apoptosis and inflammatory injury of microglia cells following spinal cord injury

2021 ◽  
Vol 16 (1) ◽  
Author(s):  
Sheng-Yu Cui ◽  
Wei Zhang ◽  
Zhi-Ming Cui ◽  
Hong Yi ◽  
Da-Wei Xu ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Spinal Cord Injury ◽  
Cell Viability ◽  
Microglial Cells ◽  
Protective Effects ◽  
Loss Of Function ◽  
Sprague Dawley ◽  
Cord Injury ◽  
Apoptosis And Inflammation

Abstract Background Spinal cord injury (SCI) is associated with health burden both at personal and societal levels. Recent assessments on the role of lncRNAs in SCI regulation have matured. Therefore, to comprehensively explore the function of lncRNA LEF1-AS1 in SCI, there is an urgent need to understand its occurrence and development. Methods Using in vitro experiments, we used lipopolysaccharide (LPS) to treat and establish the SCI model primarily on microglial cells. Gain- and loss of function assays of LEF1-AS1 and miR-222-5p were conducted. Cell viability and apoptosis of microglial cells were assessed via CCK8 assay and flow cytometry, respectively. Adult Sprague-Dawley (SD) rats were randomly divided into four groups: Control, SCI, sh-NC, and sh-LEF-AS1 groups. ELISA test was used to determine the expression of TNF-α and IL-6, whereas the protein level of apoptotic-related markers (Bcl-2, Bax, and cleaved caspase-3) was assessed using Western blot technique. Results We revealed that LncRNA LEF1-AS1 was distinctly upregulated, whereas miR-222-5p was significantly downregulated in LPS-treated SCI and microglial cells. However, LEF1-AS1 knockdown enhanced cell viability, inhibited apoptosis, as well as inflammation of LPS-mediated microglial cells. On the contrary, miR-222-5p upregulation decreased cell viability, promoted apoptosis, and inflammation of microglial cells. Mechanistically, LEF1-AS1 served as a competitive endogenous RNA (ceRNA) by sponging miR-222-5p, targeting RAMP3. RAMP3 overexpression attenuated LEF1-AS1-mediated protective effects on LPS-mediated microglial cells from apoptosis and inflammation. Conclusion In summary, these findings ascertain that knockdown of LEF1-AS1 impedes SCI progression via the miR-222-5p/RAMP3 axis.

scholarly journals Lipopolysaccharide induces neuroinflammation in microglia by activating the MTOR pathway and downregulating Vps34 to inhibit autophagosome formation

2020 ◽  
Vol 17 (1) ◽  
Author(s):  
Xiaoxia Ye ◽  
Mingming Zhu ◽  
Xiaohang Che ◽  
Huiyang Wang ◽  
Xing-Jie Liang ◽  
...  
Keyword(s):  
Inflammatory Response ◽  
Microglial Activation ◽  
Adaptor Protein ◽  
Mtor Pathway ◽  
Microglial Cells ◽  
Inflammatory Factors ◽  
Intraventricular Injection ◽  
Enzyme Linked Immunosorbent Assay ◽  
Autophagic Flux

Abstract Background Microglial activation is a prominent feature of neuroinflammation, which is present in almost all neurodegenerative diseases. While an initial inflammatory response mediated by microglia is considered to be protective, excessive pro-inflammatory response of microglia contributes to the pathogenesis of neurodegeneration. Although autophagy is involved in the suppression of inflammation, its role and mechanism in microglia are unclear. Methods In the present study, we studied the mechanism by which lipopolysaccharide (LPS) affects microglial autophagy and the effects of autophagy on the production of pro-inflammatory factors in microglial cells by western blotting, immunocytochemistry, transfection, transmission electron microscopy (TEM), and real-time PCR. In a mouse model of neuroinflammation, generated by intraventricular injection of LPS (5 μg/animal), we induced autophagy by rapamycin injection and investigated the effects of enhanced autophagy on microglial activation by enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry. Results We found that autophagic flux was suppressed in LPS-stimulated N9 microglial cells, as evidenced by decreased expression of the autophagy marker LC3-II (lipidated form of MAP1LC3), as well as increased levels of the autophagy adaptor protein SQSTM1. LPS significantly decreased Vps34 expression in N9 microglial cells by activating the PI3KI/AKT/MTOR pathway without affecting the levels of lysosome-associated proteins and enzymes. More importantly, overexpression of Vps34 significantly enhanced the autophagic flux and decreased the accumulation of SQSTM1 in LPS-stimulated N9 microglial cells. Moreover, our results revealed that an LPS-induced reduction in the level of Vps34 prevented the maturation of omegasomes to phagophores. Furthermore, LPS-induced neuroinflammation was significantly ameliorated by treatment with the autophagy inducer rapamycin both in vitro and in vivo. Conclusions These data reveal that LPS-induced neuroinflammation in N9 microglial cells is associated with the inhibition of autophagic flux through the activation of the PI3KI/AKT/MTOR pathway, while enhanced microglial autophagy downregulates LPS-induced neuroinflammation. Thus, this study suggests that promoting the early stages of autophagy might be a potential therapeutic approach for neuroinflammation-associated diseases.

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