Apoptosis In Astrocytes Research Articles

Effect of Sirtuin-1 and Wnt/β-Catenin Signaling Pathway in Rat Model of Spinal Cord Injury.

Sirtuin-1 (SIRT1) has anti-inflammatory and antioxidant effects and has been reported to be involved in spinal cord injury (SCI). Wnt/β-catenin signal has been shown to play a critical role in the pathogenesis of chronic diseases, and it participated in the recovery of nerve function after SCI. However, the specific link between them in SCI is unclear. In addition, targeting posttraumatic astrocyte apoptosis is crucial for improving neural degeneration and locomotor function. Therefore, in this article, we studied the relationship of β-catenin and SIRT1 using in the SCI rat model and primary astrocyte treated with hydrogen peroxide (H2O2) or lithium chloride (LiCl). Results showed that after SCI, SCI area and motor function recover over time, and β-catenin is gradually increased to the seventh day and then in turn decreases until 4 weeks, positively correlated with cell apoptosis. The expression of SIRT1 and downstream FOXO4 gradually increased, and β-catenin is negatively correlated with SIRT1 expression. Moreover, treatment with H2O2 in primary cultured astrocyte significantly increased β-catenin and Caspase-3 expression, while decreased SIRT1 and Forkhead box O- (FOXO-) 4. The immunofluorescence results are consistent with this. Administration of LiCl further aggravates the above results. These findings suggest that SIRT1 is negatively correlated with β-catenin in SCI, which promotes the apoptosis of motor neuron cells, which may be related to the participation of FOXO4.

Post-ischemia common carotid artery occlusion worsens memory loss, but not sensorimotor deficits, in long-term survived stroke mice

Ischemic stroke in rodents is usually induced by intraluminal occlusion of the middle cerebral artery (MCA) via the external carotid artery (ECA) or the common carotid artery (CCA). The latter route requires permanent CCA occlusion after ischemia, and here, we assess its effects on long-term outcomes. Transient occlusion of MCA and CCA was performed at normal body temperature. After 90 min of ischemia, mice were randomized to permanent CCA occlusion or no occlusion (control group). Body weight, and motor and sensory functions, ie, pole test, adhesive tape removal, and elevated plus maze, were evaluated at 24 h, and at 7 and 28 days after stroke. Infarct volume, apoptosis, and activation of astrocytes and microglia were assessed at 4 weeks by an investigator blinded to groups. The Morris water maze test was performed at 3 weeks in the second experiment. One mouse died at 4 days, and the other mice survived with persistent neurologic deficits. CCA-occluded mice exhibited delayed turn on the pole at 24 h and decreased responses to the von Frey filament, and spent more time on the pole at 7 and 28 days than the control group. Infarction, hemispheric atrophy, glial activation, and apoptotic neuronal death were present in all mice, and no intra-group difference was found. However, CCA-occluded mice had a significantly poorer performance in the Morris water maze compared to the control group, which showed an adverse effect of post-ischemia CCA occlusion on cognition. Thus, the model selection should be well considered in preclinical efficacy studies on stroke-induced vascular dementia and stroke with Alzheimer’s disease.

Astrocytic MicroRNA in Ageing, Inflammation, and Neurodegenerative Disease.

Astrocytes actively regulate numerous cell types both within and outside of the central nervous system in health and disease. Indeed, astrocyte morphology, gene expression and function, alongside the content of astrocyte-derived extracellular vesicles (ADEVs), is significantly altered by ageing, inflammatory processes and in neurodegenerative diseases, such as Alzheimer’s disease, Parkinson’s disease and amyotrophic lateral sclerosis. Here, we review the relevant emerging literature focussed on perturbation in expression of microRNA (miRNA), small non-coding RNAs that potently regulate gene expression. Synthesis of this literature shows that ageing-related processes, neurodegenerative disease-associated mutations or peptides and cytokines induce dysregulated expression of miRNA in astrocytes and in some cases can lead to selective incorporation of miRNA into ADEVs. Analysis of the miRNA targets shows that the resulting downstream consequences of alterations to levels of miRNA include release of cytokines, chronic activation of the immune response, increased apoptosis, and compromised cellular functioning of both astrocytes and ADEV-ingesting cells. We conclude that perturbation of these functions likely exacerbates mechanisms leading to neuropathology and ultimately contributes to the cognitive or motor symptoms of neurodegenerative diseases. This field requires comprehensive miRNA expression profiling of both astrocytes and ADEVs to fully understand the effect of perturbed astrocytic miRNA expression in ageing and neurodegenerative disease.

The association of Edaravone with shunt surgery improves behavioral performance, reduces astrocyte reaction and apoptosis, and promotes neuroprotection in young hydrocephalic rats

The neuroprotective effect of Edaravone in young hydrocephalic rats associated with a CSF derivation system was evaluated. The drug has already been shown to be beneficial in experimental hydrocephalus, but the combination of this drug with shunt surgery has not yet been investigated. Fifty-seven-day-old Wistar rats submitted to hydrocephalus by injection of kaolin in the cisterna magna were used and divided into five groups: control (n = 10), hydrocephalic (n = 10), hydrocephalic treated with Edaravone (20 mg/kg/day) (n = 10), hydrocephalic treated with shunt (n = 10) and hydrocephalic treated with shunt and Edaravone (n = 10). Administration of the Edaravone was started 24 h after hydrocephalus induction (P1) and continued until the experimental endpoint (P21). The CSF shunt surgery was performed seven days after hydrocephalus induction (P7). Open-field tests, histological evaluation by hematoxylin and eosin, immunohistochemistry by Caspase-3 and GFAP, and ELISA biochemistry by GFAP were performed. Edaravone reduced reactive astrogliosis in the corpus callosum and germinal matrix (p < 0.05). When used alone or associated with CSF shunt surgery, the drug decreased the cell death process (p < 0.0001) and improved the morphological aspect of the astroglia (p < 0.05). The results showed that Edaravone associated with CSF bypass surgery promotes neuroprotection in young hydrocephalic rats by reducing reactive astrogliosis and decreasing cell death.

The effects of gabapentin and ethanol on the regulated cell death of astrocytes in primary culture

Purpose: In this study, the effect of gabapentin on the regulated cell death of astrocytes in primary culture was examined. Because astrocytes are relatively resistant to decay by apoptotic pathways, the effect of different concentrations of gabapentin on apoptosis in necroptosis was tested as another form of regulatedcell death. In addition, the impact of gabapentin on the death of astrocytes that were exposed to ethanol was also examined.&#x0D; Methods: Primary cultures of astrocytes that were obtained from the brain cortex of newborn rats were used as the experimental model. Cells were exposed to different concentrations of gabapentin only, ethanol only or to a combination of ethanol and gabapentin. Using flow cytometry, the proportions of viable, early apoptotic, necroptotic, and secondary dead cells were determined.&#x0D; Results: The effect of gabapentin on early astrocytic apoptosis and necroptosis was dependent on concentration. In concentrations of up to 10 μg/mL, gabapentin did not affect astrocyte deaths; whereas at higher concentrations, the proportion of necroptotic cells increased. The concomitant exposure of the cells to gabapentin (10 μg/mL) and ethanol (100 mM) for 24 hours did not significantly affect cell death caused by ethanol. For cells that are exposed to 50 mM ethanol for 7 days, gabapentin slightly reduced the proportion of necrotic cells.&#x0D; Conclusion: Gabapentin did not affect the viability of astrocytes in concentrations up to 10 μg/mL. The concomitant exposure of astrocytes to ethanol and gabapentin for 24 hours did not reduce the toxicity of ethanol. In astrocytes that are chronically exposed to ethanol, gabapentin slightly reduced the effect of ethanol on necroptosis.

Dexmedetomidine attenuates propofol-induced apoptosis of neonatal hippocampal astrocytes by inhibiting the Bcl2l1 signalling pathway.

Apoptosis shapes brain structure and function during early life. However, aberrant apoptosis, including that associated with the general anaesthetic propofol, is undesirable. Dexmedetomidine (DEX) provides potential neuroprotection against apoptosis, but the underlying mechanism remains unknown. We exposed neonatal rodent hippocampal astrocytes to propofol alone and in combination with DEX and yohimbine (an α2 -adrenergic receptor antagonist), then evaluated cell viability using the MTT assay. The underlying regulatory mechanism associated with apoptosis-related genes was detected using a combinational strategy including double immunofluorescent staining, real-time reverse transcription polymerase chain reaction (RT-PCR), western blot, and flow cytometry. Propofol reduced matrix metallopeptidase 9 (MMP9) in cultured astrocytes, and activated the rno-miR-665/Bcl2-like 1 (Bcl2l1)/cleaved caspase 9 (CC9)/cleaved caspase 3 (CC3) pathway. Combinations incorporating propofol with A-1155463 (a selective Bcl2l1 inhibitor) or MMP9 antagomir reduced Bcl2l1 and promoted apoptosis. Co-culture of propofol with Bcl2l1 or with MMP9 agomir was sufficient to decrease the pro-apoptotic effects of propofol. Interestingly, DEX alone had no significant effect on apoptosis. When combined with propofol, however, the negative effects of propofol on the MMP9 and apoptosis-related genes (Bcl2l1, CC9, and CC3) were reduced. Furthermore, yohimbine pretreatment blocked the neuroprotective effects of DEX. Rno-miR-665 was also found to reduce MMP9 expression in propofol-treated hippocampal astrocytes. Taken together, the results indicate that DEX pretreatment reduces propofol-associated pro-apoptosis in developing astrocytes via downregulation of anti-apoptotic signalling mediated by Bcl2l1.

HIV infection of astrocytes compromises inter-organelle interactions and inositol phosphate metabolism: A potential mechanism of bystander damage and viral reservoir survival.

HIV-associated neurological dysfunction is observed in more than half of the HIV-infected population, even in the current antiretroviral era. The mechanisms by which HIV mediates CNS dysfunction are not well understood but have been associated with the presence of long-lasting HIV reservoirs. In the CNS, macrophage/microglia and a small population of astrocytes harbor the virus. However, the low number of HIV-infected cells does not correlate with the high degree of damage, suggesting that mechanisms of damage amplification may be involved. Here, we demonstrate that the survival mechanism of HIV-infected cells and the apoptosis of surrounding uninfected cells is regulated by inter-organelle interactions among the mitochondria/Golgi/endoplasmic reticulum system and the associated signaling mediated by IP3 and calcium. We identified that latently HIV-infected astrocytes had elevated intracellular levels of IP3, a master regulator second messenger, which diffuses via gap junctions into neighboring uninfected astrocytes resulting in their apoptosis. In addition, using laser capture microdissection, we confirmed that bystander apoptosis of uninfected astrocytes and the survival of HIV-infected astrocytes were dependent on mitochondrial function, intracellular calcium, and IP3 signaling. Blocking gap junction channels did not prevent an increase in IP3 or inter-organelle dysfunction in HIV-infected cells but reduced the amplification of apoptosis into uninfected neighboring cells. Our data provide a mechanistic explanation for bystander damage induced by surviving infected cells that serve as viral reservoirs and provide potential targets for interventions to reduce the devastating consequences of HIV within the brain.

MicroRNA-367-3p suppresses sevoflurane-induced adult rat astrocyte apoptosis by targeting BCL2L11.

The present study aimed to characterize the effect of microRNA (miR)-367-3p on sevoflurane anesthesia and elucidate the underlying mechanism. A total of 36 4-month-old adult Sprague-Dawley rats were divided into six groups. Sevoflurane was inhaled at concentrations of 0, 1, 2, 4, 8 and 16% for a total of 6 h; the hippocampus of the brain was subsequently minced and digested, and astrocytes were isolated. Various methods, including reverse transcription-quantitative (RT-q)PCR, western blotting and TUNEL staining, were used to determine the expression levels of Bax, BCL-2 and BCL-2-like protein 11 (BCL2L11), as well as the level of apoptosis. The rats were treated with 8% sevoflurane and the astrocytes from the rats were transfected with miR-367-3p or anti-miR-367-3p. The present study demonstrated that sevoflurane promoted astrocytes apoptosis. Western blotting revealed that with an increase of sevoflurane concentration, the expression levels of the apoptotic proteins Bax and BCL2L11 were significantly increased, whereas the protein expression levels of BCL-2 were significantly decreased. However, overexpression of miR-367-3p reversed these effects. TUNEL staining revealed that sevoflurane promoted the apoptosis of astrocytes, while apoptosis was reversed by miR-367-3p overexpression. RT-qPCR demonstrated that sevoflurane inhibited the expression of miR-367-3p. Notably, miR-367-3p reduced the expression of BCL2L11, thereby inhibiting the apoptosis of astrocytes originating from the hippocampal area of adult rats induced by sevoflurane. Therefore, miR-367-3p and BCL2L11 may act as effective targets for the study of anesthesia.

Downregulation of MicroRNA-145-5p in Activated Microglial Exosomes Promotes Astrocyte Proliferation by Removal of Smad3 Inhibition.

In spinal cord injury, microglial activation plays an important role during the inflammatory process. Specifically, the cellular and molecular interactions between microglia and astrocytes are of critical importance. Cells can communicate with each other through the substances carried by exosomes, and overproliferated astrocytes would create a physical and chemical barrier that prevents neurite regeneration, thereby interfering with functional recovery. On the other hand, Smad3 is an important factor in the proliferation, migration, and apoptosis of astrocytes. In this study, supernatant and purified exosomes were collected from LPS-treated microglia and co-cultured with astrocytes. The results showed that astrocytic proliferation was promoted with higher levels of Smad3. Furthermore, miRNA sequencing analysis was performed on microglial exosomes after inflammation. The results revealed a differential expression of miR-145-5p in the exosomes. The Dual-Luciferase assay showed that miR-145-5p could bind to Smad3 mRNA and regulate the levels of Smad3 protein at the post-transcriptional level. Subsequently, exosomes were transfected with miR-145-5p mimics, and astrocytes after mechanical injury were cultured with these exosomes for 24h. The levels of Smad3 and phosphor-Smad3 proteins were analyzed by western blot and qRT-PCR. CCK8 and flow cytometry showed lower proliferation of astrocytes after co-culturing with the exosomes transfected with the miR-145-5p mimic. This study finds that miR-145-5p was found to be a negative regulator of astrocyte proliferation, and that its downregulation promotes smad3 activity and thus astrocyte proliferation.

1,2-Dichloroethane induces apoptosis in the cerebral cortexes of NIH Swiss mice through microRNA-182-5p targeting phospholipase D1 via a mitochondria-dependent pathway

1,2-Dichloroethane (1,2-DCE) is a pervasive environmental pollutant found in ambient and residential air, as well as ground and drinking water. Overexposure to it results in cortex edema, in both animals and humans. 1,2-DCE induces apoptosis in the cerebellum, liver and testes. This promotes the hypothesis that 1,2-DCE may induce apoptosis in the cortex as brain edema progresses. To validate our hypothesis, 40 NIH male mice were exposed to 0, 100, 350, 700 mg/m3 1,2-DCE by whole-body dynamic inhalation for 28 consecutive days. MicroRNA (miRNA) and mRNA microarray combined with TdT-mediated dUTP nick-end labeling, flow cytometry, and mitochondrial membrane potential (mtΔΨ) measurement were applied to identify the cortex apoptosis pathways' specific responses to 1,2-DCE, in vitro and in vivo. The results showed that 1,2-DCE caused brain edema and increased apoptosis in the mouse cortexes. We confirmed that 1,2-DCE induced increased apoptosis via mitochondrial pathway, both in vitro and in vivo, as evidenced by increased Caspase-3, cleaved Caspase-3, Cytochrome c and Bax expression, and decreased Bcl-2 expression. Additionally, mtΔΨ decreased after 1,2-DCE treatment in vitro. 1,2-DCE exposure increased miR-182-5p and decreased phospholipase D1 (PLD1) in the cerebral cortex of mice. MiR-182-5p overexpression and PLD1 inhibition reduced mtΔΨ and increased astrocyte apoptosis, yet miR-182-5p inhibition alleviated the 1,2-DCE-induced PLD1 down-regulation and the increased apoptosis. Finally, PLD1 was confirmed to be a target of miR-182-5p by luciferase assay. Taken together, our findings indicate that 1,2-DCE exposure induces apoptosis in the cortex via a mitochondria-dependent pathway. This pathway is regulated by a miR-182-5p⊣PLD1 axie.

Inhibit inflammation and apoptosis of pyrroloquinoline on spinal cord injury in rat.

BackgroundPyrroloquinoline quinone (PQQ) is a redox cofactor that can participate in a variety of physiological and biochemical processes, such as anti-inflammatory, cytoprotection, anti-aging, and anti-apoptosis. PQQ plays an important protective role in the central nervous system (CNS). However, the effects of PQQ on astrocytes of the CNS and spinal cord injury (SCI) of rats is still unclear. The present study investigates the role of PQQ in inflammation, apoptosis, and autophagy after SCI in rats. And the effect of PQQ on lipopolysaccharide (LPS)-induced apoptosis and inflammation of astrocytes in vitro, to explore the neuroprotective mechanism of PQQ.MethodsSixty specific pathogen free (SPF) SD male rats (200–250 g) were randomly divided into Normal group, Sham group, SCI group, and SCI + PQQ group, with 15 rats in each group. BBB score, HE staining, Nissl staining, Western blot, immunofluorescence, and other methods were used for detection.ResultsOur results showed that PQQ could upregulate BBB score in SCI rats. In the second place, PQQ can increase the number and improve the morphology of neurons after SCI. The expression of IL-1β, TNF-α, IL-6 was significantly decreased after PQQ treatment. And then, the ratio of B-cell lymphoma-2 (Bcl-2)/Bcl-2 associated X protein (Bax) increased significantly, and the positive signal of NeuN increased obviously after PQQ treatment. There are a large number of co-localizations between Bcl-2 and NeuN. Meanwhile, PQQ could down-regulate the expression of Active-Caspase3, and PQQ treatment could reverse the transfer of Active-Caspase3/Caspase3 from the cytoplasm to the nucleus in neurons and astrocytes after SCI. At the same time, PQQ had no significant effect on the LC3b/a ratio. PQQ could decrease the LAMP2 expression in spinal cord after injury. The expression level of phospho-Akt (p-AKT) increased after SCI and decreased after PQQ treatment. In primary astrocytes, LPS could induce the expression levels of IL-1β, TNF-α, and IL-6, and which were inhibited by PQQ treatment at 12 hours. After treatment with LPS, the expression level of Active-Caspase3 increased, which could be reversed by PQQ treatment for 24 h.ConclusionsThese results suggest that PQQ can ameliorate the motor function of hind limbs and the pathological changes of neurons and injured spinal cord after SCI, down-regulate the expressions of IL-1β, TNF-α, and IL-6, inhibit apoptosis after SCI, and inhibit LPS-induced apoptosis and inflammation of astrocytes.

Circular RNA 0025984 Ameliorates Ischemic Stroke Injury and Protects Astrocytes Through miR-143-3p/TET1/ORP150 Pathway.

MiR-143-3p is aberrantly expressed in patients with ischemic stroke and associated with ischemic brain injury. However, the underlying mechanisms are largely unknown. Here, we confirmed circ_0025984 and TET1 as a sponge and target of miR-143-3p, respectively, by luciferase reporter assay. In astrocytes, OGD significantly decreased circ_0025984 and TET1 levels but increased miR-143-3p levels, which was also observed in brains of mice with MCAO. Treatment with miR-143-3p inhibitor or circ_0025984 significantly decreased astrocyte apoptosis and autophagy, as well as cerebral injury and neuron loss in mice with MCAO. Notably, TET1 overexpression decreased astrocyte apoptosis and autophagy and induced promoter hypomethylation and expression of ORP150. Our results demonstrated for the first time that circ_0025984 protects astrocytes from ischemia-induced autophagy and apoptosis by targeting the miR-143-3p/TET1 pathway and might inhibit cerebral injury induced by ischemic stroke. Furthermore, our data revealed the important positive regulation of ORP150 by TET1, which could be associated with its neuroprotective role. Graphical abstract Model for signaling pathway of circ_0025984/miR-143-3p/TET1 inastrocytes cultured under OGD. In astrocytes, circ_0025984 acts as a sponge of miR-143-3p, which directly targets TET1 and decreases its expression (A). After translocatinginto the nucleus, TET1 binds to the promoter of ORP150, converts 5mC into 5hmC,leading to DNA demethylation and increased expression of ORP150 (B). In astrocytescultured under OGD, ER stress is induced and eventually leads to apoptosis andautophagy mediated by ATG7, which is regulated by circ_0025984 via ORP150 andGRP78 (C).

β-HB inhibits the apoptosis of high glucose-treated astrocytes via activation of CREB/BDNF axis

ABSTRACT Objective Nerve damage can cause severe limb dysfunction and even leave a lifelong disability. The apoptosis of astrocytes may contribute to the nerve damage. In this research, we sought to investigate the effect of β-HB on nerve damage in vitro. Design Astrocytes were treated with high glucose (HG) to mimic in vitro model of nerve damage. RT-qPCR and western blot were used to detect expressions of CREB, BDNF, Ki-67, PCNA, Bax, Bcl-2 and cleaved caspase 3 in astrocytes, respectively. MTT was used to measure the cell viability. In addition, flow cytometry was used to detect the cell apoptosis. Results β-HB significantly promoted the proliferation and inhibited apoptosis in HG-treated astrocytes. Results showed that of PCNA and Bcl-2 were upregulated, and Bax and cleaved caspase 3 were downregulated after β-HB stimulated in HG-treated astrocytes. In addition, HG-induced inhibition on BDNF expression in astrocytes was notably reversed by β-HB. Furthermore, β-HB promoted the growth and inhibited apoptosis of high glucose-treated astrocytes via activation of CREB/BDNF axis. Conclusion β-HB promotes the growth and inhibits the apoptosis of high glucose-treated astrocytes via activation of CREB/BDNF axis, which may serve as a new target for treatment of nerve damage.

Overexpression of miR-221-3p effecting cell proliferation, apoptosis and inflammation by targeting Toll-like receptors 4 in propofol-induced rat astrocytes

IntroductionGrowing evidence indicated that propofol has neurotoxic effects on the brains of developing rodents, leading to neuronal cell death, neurodegeneration, and brain injury.Material and methodsEctopic miR-221-3p was transfected into rat astrocytes, and Cell Counting Kit-8 assay and flow cytometry were performed to evaluate cell growth and apoptosis. The mRNA levels of Toll-like receptors 4 (TLR4), nuclear factor kappa B, interleukin-6, interleukin-1β, myeloid differentiation primary response 88 (MyD88), caspase-3, caspase-12, STAT3, and GRP78 were detected using quantitative real-time polymerase chain reaction. The proteins of TLR4 and MyD88 were determined using Western blotting. The association between miR-221-3p and TLR4 was measured using Dual-Luciferase Reporter Assay (Promega Corporation, Wisconsin, USA). Then, siTLR4 was transfected with 293T cells to study the role of TLR4 in astrocytes with propofol treatment.ResultsThe miR-221-3p expression in rat astrocytes was markedly suppressed by propofol treatment. The miR-221-3p mimics transfection in propofol-treated astrocytes effectively reduced the suppressive effect of propofol on astrocyte growth, repressed the propofol-induced apoptosis in rat astrocytes, and decreased the cell number during the G2–M phase. The expression of MyD88 and TLR4 was induced by propofol, whereas the transfection of miR-221-3p mimics dramatically reduced these genes expression at the mRNA and protein expression. After that, TLR4 was found to be target of miR-221-3p using Dual-Luciferase Reporter Assay. Furthermore, knockdown of TLR4 could suppress the apoptosis rate in propofol-treated astrocytes.ConclusionsThis study revealed that miR-221-3p might prevent astrocytes from propofol-induced damage by targeting TLR4.

Apoptotic investigation of brain tissue cells in dogs naturally infected by canine distemper virus

BackgroundCanine distemper caused by canine distemper virus that belongs to the Morbillivirus genus of the Paramyxoviridae family is still a global epidemic significant infectious disease, especially in pet dogs in China and serious harm to the development of the dog industry. It has been known that apoptosis caused by the canine distemper virus can show in culture cells, lymphoid tissues, and the cerebellum. However, its occurrence in brain tissue cells remains unclear. To investigate the relationship among canine distemper infecting brain tissues, apoptosis in brain tissue cells, and demyelinating pathogenesis was investigated.Methods16 naturally infected dogs that exhibited clinical signs of CD and tested positive for the anti-CDV monoclonal antibody and six healthy dogs that served as the control, were used in the research. Brain specimens were divided into the cerebrum, brain stem, and cerebellum embedded in paraffin and made the sections respectively. Approximately 5 µm-thick sections were stained by hematoxylin–eosin, methyl green pyronin, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling technique, and immunohistochemistry. CDV nucleocapsid protein was detected by immune streptavidin-biotinylated peroxidase complex.ResultsAlterations in the brain tissues of CDV-infected dogs involved both various cells and nerve fibers. CDV had varying degrees of cytotropism to all brain tissue cells; apoptosis also occurred in all brain cells, especially in the endothelia of cerebral vessels, astrocytes, oligodendrocytes, and ependymal cells, the more serious infection, the more obvious apoptosis. Serious infections also involved the pyramidal and Purkinje cells. The nervous fibers exhibited demyelinating lesions (showed small multifocal vacuole), and some axonal neuron atrophy gradually disappeared (formed large vacuole).ConclusionsApoptosis in brain tissue cells was mainly related to the propagation path and cytotropism of CDV. The apoptosis of astrocytes, oligodendrocytes, and some neurons may play a significant role in the demyelinating pathogenesis in dogs with acute canine distemper. A lot of diverse nervous signs shown in the clinic may be related to different neuron apoptosis.

Astrocyte Pannexin 1 Suppresses LPS-Induced Inflammatory Responses to Protect Neuronal SH-SY5Y Cells.

Reactive astrogliosis is a key hallmark of inflammatory responses in the pathogenesis of brain injury, including Parkinson’s disease (PD), but its role and regulatory mechanisms are not fully understood. Pannexin 1 (Panx 1) is a membrane channel that mediates substance release in many neurodegenerative diseases. However, the role of astrocyte Panx 1 in the regulation of PD-like neuroinflammation remains elusive. Here, we characterized the expression of Panx 1 in isolated primary astrocytes and a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD model. The functions of Panx 1 in inflammatory cytokines expression and the viability of neuronal SH-SY5Y cells were examined in cultured cells treated with lipopolysaccharide (LPS) and 1-methyl-4-phenylpyridinium (MPP+). We found that Panx 1 expression was significantly increased under both LPS- and MPP+-treated conditions. Panx 1 downregulation suppressed LPS-induced pro-inflammatory cytokine expression but did not significantly affect MPP+-induced astrocyte apoptosis or inflammatory cytokine expression through treatment with the Panx 1 inhibitor carbenoxolone (CBX) and Panx 1 siRNA. Moreover, silencing Panx 1 in reactive astrocytes had a potentially protective effect on the viability of neuronal SH-SY5Y cells. Therefore, we propose that Panx 1 may serve as a key regulator in reactive astrocytes to intervene in the inflammatory response and maintain neuronal viability in the context of PD-like conditions.

Orexin-A alleviates astrocytic apoptosis and inflammation via inhibiting OX1R-mediated NF-κB and MAPK signaling pathways in cerebral ischemia/reperfusion injury

Orexin-A (OXA) is a neuropeptide with neuroprotective effect by reducing cerebral ischemia/reperfusion injury (CIRI). Inflammation and apoptosis mediated by astrocyte activation are the key pathological mechanisms for CIRI. We thus attempted to confirm neuroprotective effects of OXA on astrocytic inflammation and apoptosis in CIRI and clarify the relative mechanisms. A middle cerebral artery occlusion and reperfusion (MCAO/R) rat model and U251 glioma cells model subjected to oxygen glucose deprivation and reperfusion (OGD/R) were established, with or without OXA treatment. Neurological deficit score was determined, and cerebral infarct volume was evaluated by 2,3,5-triphenyltetrazolium chloride (TTC) staining. Western Blot was used to detect the expressions of NF-κB p65, p-p65, p-ERK, p-p38, GFAP, OX1R, IL-1β, TNF-α, IL-6, iNOS, Bcl-2, Bax, CytC, cleaved caspase-9 and cleaved caspase-3 in vivo and in vitro. Pro-inflammatory cytokines in cell supernatant IL-1β, TNF-α and IL-6 were determined by ELISA. Hoechst 33342 staining was used to detect the apoptosis of astrocyte. Immunofluorescent staining was performed to assess the nuclear translocation of p65 and the expression of GFAP. The results showed that OXA significantly improved neurological deficit score and decreased the volume of infarct area in brain. OXA decreased inflammatory mediators, inhibited astrocyte activation and nuclear translocation of NF-κB and phosphorylation of NF-κB, MAPK/ERK and MAPK/p38. Besides, OXA suppressed apoptosis via upregulating the ratio of Bcl-2/Bax and downregulating cytochrome C, cleaved-caspase-9 and cleaved caspase-3. Overall, it was concluded that OXA exerts neuroprotective effect during CIRI through attenuating astrocytes apoptosis, astrocytes activation and pro-inflammatory cytokines production, by Inhibiting OX1R-mediated NF-κB, MAPK/ERK and MAPK/p38 signaling pathways. The progress in our study is helpful to elucidate the molecular mechanisms of OXA neuroprotection, which could lead to the development of new treatment strategies for ischemic stroke.

Folic Acid Inhibits Aging-Induced Telomere Attrition and Apoptosis in Astrocytes In Vivo and In Vitro.

Folic acid (FA) has been reported to inhibit astrocyte apoptosis and improve aging-induced disorders; however, its role in telomere attrition remains unclear. In present study, 4-month-old senescence-accelerated mouse prone 8 (SAMP8) mice were assigned to four treatment groups for the in vivo experiment: FA-deficient diet (FA-D) group, FA-normal diet (FA-N) group, low FA-supplemented diet (FA-L) group, and high FA-supplemented diet (FA-H) group. These mice were euthanized when 10months old. There was also a young SAMP8 (4 months old) control group (Con-Y) fed with FA-normal diet. In in vitro study, primary cultures of astrocytes from hippocampus and cerebral cortex were incubated for five generations with various concentrations of FA (0-40μM) and were assigned to five groups: FA 0μM (generation 5), FA 10μM (generation 5), FA 20μM (generation 5), FA 40μM (generation 5), and FA 10μM (generation 1). The results showed that FA supplementation inhibited aging-induced astrocytosis, astrocyte apoptosis, neurodegeneration, and prevented telomere attrition in hippocampus and cortex of SAMP8 mice. FA supplementation also decreased apoptosis and telomere attrition, and increased telomerase activity, in primary cultures of astrocytes. These results showed that it may be one of the mechanisms that FA inhibiting aging-induced apoptosis of astrocyte by alleviating telomere attrition.

Acrylamide Induces Abnormal mtDNA Expression by Causing Mitochondrial ROS Accumulation, Biogenesis, and Dynamics Disorders.

Acrylamide, a well-documented neurotoxicant, is commonly found as a byproduct of the Maillard reaction in carbohydrate-rich foods. Numerous studies have indicated that acrylamide-induced apoptosis accompanied by mitochondrial dysfunction contributes to its neurotoxicity. However, the mechanisms of how acrylamide causes mitochondrial impairment is not well understood. In this study, we observed destroyed redox balance, accumulated mitochondrial reactive oxygen species (ROS), damaged mitochondrial structures, and activated apoptosis in astrocytes following acrylamide treatment. Furthermore, acrylamide decreased the expression of mitochondrial biogenesis- and dynamics-related genes, including PGC-1α, TFAM, Mfn2, and Opa1, and altered the expression of mitochondrial DNA (mtDNA)-encoded mitochondrial respiratory chain complexes, along with the inhibited mitochondrial respiration. Pretreatment with a mitochondrial ROS scavenger mitoquinone dramatically restored the expressions of PGC-1α, TFAM, Mfn2, and Opa1; protected the mitochondrial structure; and decreased acrylamide-induced apoptosis. Further in vivo experiments confirmed that acrylamide decreased the expressions of PGC-1α, TFAM, Mfn2, and Opa1 in rat brain tissues. These results revealed that acrylamide triggered the mitochondrial ROS accumulation to interfere with mitochondrial biogenesis and dynamics, causing mtDNA damage and finally resulting in mitochondrial dysfunction and apoptosis.

BMSCs Regulate Astrocytes through TSG-6 to Protect the Blood-Brain Barrier after Subarachnoid Hemorrhage.

Background In patients with subarachnoid hemorrhage (SAH), the damage of the blood-brain barrier (BBB) can be life-threatening. Mesenchymal stem cells are widely used in clinical research due to their pleiotropic properties. This study is aimed at exploring the effect of BMSCs regulating astrocytes on the BBB after SAH. Methods The SAH model was established by perforating the blood vessels. BMSCs were transfected with TSG-6 inhibitor plasmid and cocultured with astrocytes. Intravenous transplantation of BMSCs was utilized to treat SAH rats. We performed ELISA, neurological scoring, Evans blue staining, NO measurement, immunofluorescence, BBB permeability, Western blot, HE staining, Nissl staining, and immunohistochemistry to evaluate the effect of BMSCs on astrocytes and BBB. Results SAH rats showed BBB injury, increased BBB permeability, and brain histological damage. BMSCs will secrete TSG-6 after being activated by TNF-α. Under the influence of TSG-6, the NF-κB and MAPK signaling pathways of astrocytes were inhibited. The expression of iNOS was reduced, while occludin, claudin 3, and ZO-1 expression was increased. The production of harmful substances NO and ONOO− decreased. The level of inflammatory factors decreased. The apoptosis of astrocytes was weakened. TSG-6 secreted by BMSCs can relieve inflammation caused by SAH injury. The increase in BBB permeability of SAH rats was further reduced and the risk of rebleeding was reduced. Conclusion BMSCs can regulate the activation of astrocytes through secreting TSG-6 in vivo and in vitro to protect BBB.