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1.
J Toxicol Sci ; 45(9): 559-567, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32879255

RESUMO

Lead is a main threat to human health due to its neurotoxicity and the astrocyte is known to be a common deposit site of lead in vivo. However, the detailed mechanisms related to lead exposure in the astrocytes were unclear. In order to deeply investigate this issue, we used Sprague-Dawley (SD) rats and astrocytes isolated from the hippocampus of SD rats to establish the lead-exposed animal and cell models through treating with lead acetate. The expression levels of GFAP, LC3, and p62 in the rat hippocampus were detected by immunofluorescence and Western blot after lead exposure. The effects of autophagy on lead-exposed astrocytes were studied by further autophagy inhibitor 3-methyladenine (3-MA) induction. Transmission electron microscopy was used to observe autophagosomes in astrocytes after lead acetate treatment, followed by assessing related autophagy protein markers. In addition, some inflammatory cytokines and oxidative stress markers were also evaluated after lead exposure and 3-MA administration. We found that lead exposure induced activation of astrocytes, as evidenced by increased GFAP levels and GFAP-positive staining cells in the rat hippocampus. Moreover, lead exposure induced autophagy in astrocytes, as evidenced by increased LC3II and Beclin 1 protein levels and decreased p62 expression in both the rat hippocampus and astrocytes, and it was confirmed that this autophagy was activated through blocking the downstream Akt/target of the rapamycin (mTOR) pathway in astrocytes. Furthermore, it was shown that treatment of lead acetate increased the release of tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß), and the accumulation of malondialdehyde (MDA) and myeloperoxidase (MPO) in astrocytes, which could be alleviated by further 3-MA induction. Therefore, we conclude that lead exposure can induce the autophagy of astrocytes via blocking the Akt/mTOR pathway, leading to accelerated release of inflammatory factors and oxidative stress indicators in astrocytes.


Assuntos
Astrócitos/metabolismo , Astrócitos/fisiologia , Autofagia/efeitos dos fármacos , Autofagia/genética , Compostos Organometálicos/toxicidade , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Animais , Células Cultivadas , Proteína Glial Fibrilar Ácida/genética , Proteína Glial Fibrilar Ácida/metabolismo , Hipocampo/citologia , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Masculino , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Estresse Oxidativo/genética , Ratos Sprague-Dawley , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo
2.
Rinsho Shinkeigaku ; 60(9): 581-588, 2020 Sep 29.
Artigo em Japonês | MEDLINE | ID: mdl-32779598

RESUMO

Alexander disease (ALXDRD) is a primary astrocyte disease caused by glial fibrillary acidic protein (GFAP) gene mutation. ALXDRD had been clinically regarded as a cerebral white matter disease that affects only children for about 50 years since the initial report in 1949; however, in the early part of the 21st century, case reports of adult-onset ALXDRD with medulla and spinal cord lesions increased. Basic research on therapies to reduce abnormal GFAP accumulation, such as drug-repositioning and antisense oligonucleotide suppression, has recently been published. The accumulation of clinical data to advance understanding of natural history is essential for clinical trials expected in the future. In this review, I classified ALXDRD into two subtypes: early-onset and late-onset, and detail the clinical symptoms, imaging findings, and genetic characteristics as well as the epidemiology and historical changes in the clinical classification described in the literature. The diagnostic criteria based on Japanese ALXDRD patients that are useful in daily clinical practice are also mentioned.


Assuntos
Doença de Alexander/diagnóstico , Adolescente , Adulto , Idade de Início , Doença de Alexander/tratamento farmacológico , Doença de Alexander/genética , Doença de Alexander/fisiopatologia , Animais , Criança , Imagem de Difusão por Ressonância Magnética , Reposicionamento de Medicamentos , Proteína Glial Fibrilar Ácida/genética , Proteína Glial Fibrilar Ácida/metabolismo , Humanos , Camundongos , Pessoa de Meia-Idade , Terapia de Alvo Molecular , Mutação , Oligonucleotídeos Antissenso , Adulto Jovem
3.
PLoS One ; 15(8): e0238104, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32822415

RESUMO

PURPOSE: To delineate responses of optic nerve head astrocytes to sustained intraocular pressure (IOP) elevation in mice. METHODS: We elevated IOP for 1 day to 6 weeks by intracameral microbead injection in 4 strains of mice. Astrocyte alterations were studied by transmission electron microscopy (TEM) including immunogold molecular localization, and by laser scanning microscopy (LSM) with immunofluorescence for integrin ß1, α-dystroglycan, and glial fibrillary acidic protein (GFAP). Astrocyte proliferation and apoptosis were quantified by Ki67 and TUNEL labeling, respectively. RESULTS: Astrocytes in normal optic nerve head expressed integrin ß1 and α-dystroglycan by LSM and TEM immunogold labeling at electron dense junctional complexes that were found only on cell membrane zones bordering their basement membranes (BM) at the peripapillary sclera (PPS) and optic nerve head capillaries. At 1-3 days after IOP elevation, abnormal extracellular spaces appeared between astrocytes near PPS, and axonal vesical and mitochondrial accumulation indicated axonal transport blockade. By 1 week, abnormal spaces increased, new collagen formation occurred, and astrocytes separated from their BM, leaving cell membrane fragments. Electron dense junctional complexes separated or were absent at the BM. Astrocyte proliferation was modest during the first week, while only occasional apoptotic astrocytes were observed by TEM and TUNEL. CONCLUSIONS: Astrocytes normally exhibit junctions with their BM which are disrupted by extended IOP elevation. Responses include reorientation of cell processes, new collagen formation, and cell proliferation.


Assuntos
Astrócitos/fisiologia , Glaucoma/patologia , Nervo Óptico/fisiologia , Animais , Apoptose , Astrócitos/citologia , Astrócitos/patologia , Proliferação de Células , Modelos Animais de Doenças , Proteína Glial Fibrilar Ácida/metabolismo , Pressão Intraocular , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Nervo Óptico/citologia , Nervo Óptico/patologia
4.
Zhongguo Ying Yong Sheng Li Xue Za Zhi ; 36(1): 77-81, 2020 Jan 28.
Artigo em Chinês | MEDLINE | ID: mdl-32476377

RESUMO

OBJECTIVE: To evaluate the effects of prenatal radiation of 850~1 900 MHz mobile phone on white matter in cerebellum of adult rat offspring. METHODS: Pregnant rats were randomly divided into short term maternal radiation group, long term maternal radiation group and control group. Rats in short term and long term maternal radiation group were exposed to 6 h/d and 24 h/d mobile phone radiation during 1-17 days of pregnancy, respectively. The cerebellums of offspring rats at the age of 3 month(n=8)were taken. Cell morphology in cerebellum was studied by hematoxylin-eosin (HE) staining. The expressions of myelin basic protein (MBP), neurofilament-L (NF-L) and glial fibrillary acidic protein (GFAP) in cerebellum of rat offspring were detected by immunohistochemistry and Western blot. RESULTS: Compared to control group, the morphological changes of purkinje cells in cerebellum were obvious in rat offspring of short term and long term maternal radiation group. Compared to control group, decreased MBP and NF-L expressions and increased GFAP expression were observed in long term maternal radiation group(all P<0.05). Compared to short term radiation group, the expressions of MBP and NF-L were down-regulated (all P<0.05) and the expression of GFAP was up- regulated(P<0.05) in long term radiation group. CONCLUSION: Prenatal mobile phone radiation might lead to the damage of myelin and axon with activity of astrocytes in cerebellum of male rat offspring, which is related to the extent of radiation.


Assuntos
Telefone Celular , Cerebelo/efeitos da radiação , Radiação Eletromagnética , Efeitos Tardios da Exposição Pré-Natal , Substância Branca/efeitos da radiação , Animais , Cerebelo/patologia , Feminino , Proteína Glial Fibrilar Ácida/metabolismo , Masculino , Proteína Básica da Mielina/metabolismo , Proteínas de Neurofilamentos/metabolismo , Gravidez , Distribuição Aleatória , Ratos , Substância Branca/patologia
5.
J Toxicol Sci ; 45(5): 271-280, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32404559

RESUMO

Environmental neurotoxins such as paraquat (PQ), manganese, and 1-1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) are associated with a higher risk of Parkinson's disease (PD). These parkinsonian toxins exert certain common toxicological effects on astroglia; however, their role in the regulatory functions of astroglial secretory proteins remains unclear. In a previous study, we observed that secretogranin II (SCG2) and secretogranin III (SCG3), which are important components of the regulated secretory pathway, were elevated in PQ-activated U118 astroglia. In the current study, we used the parkinsonian toxins dopamine (DA), active metabolite of MPTP (MPP+), MnCl2, and lipopolysaccharide (LPS) as inducers, and studied the potential regulation of SCG2 and SCG3. Our results showed that all the parkinsonian toxins except LPS affected astroglial viability but did not cause apoptosis. Exposure to DA, MPP+, and MnCl2 upregulated glial fibrillary acidic protein (GFAP), a marker for astrocyte activation, and stimulated the levels of several astrocytic-derived factors. Further, DA, MPP+, and MnCl2 exposure impeded astroglial cell cycle progression. Moreover, the expression of SCG3 was elevated, while its exosecretion was inhibited in astroglia activated by parkinsonian toxins. The level of SCG2 remained unchanged. In combination with our previous findings, the results of this study indicate that SCG3 may act as a cofactor in astrocyte activation stimulated by various toxins, and the regulation of SCG3 could be involved in the toxicological mechanism by which parkinsonian toxins affect astroglia.


Assuntos
Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Cromograninas/fisiologia , Intoxicação por MPTP/complicações , Neurotoxinas/toxicidade , Doença de Parkinson Secundária/etiologia , Ciclo Celular/efeitos dos fármacos , Cloretos/efeitos adversos , Cloretos/toxicidade , Cromograninas/metabolismo , Dopamina/administração & dosagem , Dopamina/toxicidade , Proteína Glial Fibrilar Ácida/metabolismo , Humanos , Compostos de Manganês/efeitos adversos , Paraquat/toxicidade , Secretogranina II/metabolismo , Secretogranina II/fisiologia , Células Tumorais Cultivadas , Regulação para Cima/efeitos dos fármacos
6.
Invest Ophthalmol Vis Sci ; 61(5): 22, 2020 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-32413125

RESUMO

Purpose: Glial fibrillary acid protein (GFAP) and vimentin are type III intermediate filament proteins, ubiquitously expressed in retinal glial cells. Under retinal stress, both GFAP and vimentin are well-known sensitive markers for retinal gliosis. However, little is known about whether these proteins are released into the vitreous body in response to retinal gliosis or are related to the severity of retinal gliosis seen in proliferative vitreoretinopathy (PVR). Methods: Vitreous fluids were collected from 44 patients who underwent pars plana vitrectomy for macular hole (Group 1; n = 8), epiretinal membrane (Group 2; n = 8), or retinal detachment (RD) with various degrees of PVR (Group 3; n = 28). The severity of PVR was determined by cumulative scores using PVR classification. GFAP, vimentin, and total protein levels from the vitreous samples were measured. Results: Both GFAP and vimentin levels were significantly elevated in vitreous fluid from Group 3 (RD) compared with Groups 1 and 2 (P < 0.01). GFAP levels (ng/mL) were 12.4 ± 9.8, 17.5 ± 17.7, and 572.0 ± 11659.7, and vimentin levels (ng/mL) were 40.8 ± 61.9, 88.6 ± 86.8, and 3952.8 ± 8179.5 in Groups 1, 2, and 3, respectively. Total protein levels were not significantly different among the three groups. Elevated GFAP and vimentin levels in Group 3 were positively correlated with the areas of RD (P < 0.01, r = 0.53 in GFAP and P < 0.05, r = 0.46 in vimentin) and PVR scores (P < 0.05, r = 0.46 in GFAP and P < 0.00001, r = 0.76 in vimentin). Conclusions: Our data suggest that human vitreous GFAP and vimentin are protein biomarkers for PVR, and reactive gliosis may play a part in PVR formation.


Assuntos
Proteína Glial Fibrilar Ácida/metabolismo , Vimentina/metabolismo , Vitreorretinopatia Proliferativa/metabolismo , Corpo Vítreo/metabolismo , Idoso , Biomarcadores/metabolismo , Feminino , Gliose , Humanos , Masculino , Pessoa de Meia-Idade
7.
Invest Ophthalmol Vis Sci ; 61(4): 15, 2020 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-32298438

RESUMO

Purpose: Pathological neovascularization and fibrosis are common pathological changes of many retinal diseases, such as proliferative retinopathy (PR) and age-related macular degeneration (AMD). Treatment modalities for these pathological changes are limited. The purpose of the present study was to test the effects of palmitoylethanolamide (PEA), an endocannabinoid mimetic amide, on retinal neovascularization and fibrosis and to determine its molecular mechanism of action. Methods: A rat Müller cell line (rMC-1), a mouse model of oxygen-induced retinopathy (OIR), and the very-low-density lipoprotein receptor (VLDLR) knockout mouse model were used. PEA was intraperitoneally injected or orally administrated in animal models. Inflammation and profibrotic changes were evaluated by western blot analysis. Glial fibrillary acidic protein (GFAP) and peroxisome proliferator-activated receptor alpha (PPARα) were measured by RT-PCR and western blot analysis. Results: Profibrotic changes were present in OIR and Vldlr-/- retinas. PEA significantly alleviated inflammation and inhibited neovascularization in OIR and Vldlr-/- retinas and suppressed profibrotic changes in OIR and Vldlr-/- retinas. Moreover, PEA potently suppressed Müller gliosis in these retinas. In rMC-1 cells, PEA suppressed Müller gliosis, reduced inflammatory cytokines, and attenuated profibrotic changes. Further, both mRNA and protein levels of PPARα were elevated in the retina under PEA treatment, and the effects of PEA were abolished in Pparα-/- OIR mice. Conclusions: PEA reduced retinal neovascularization and fibrotic changes and suppressed Müller gliosis in experimental PR and neovascular AMD by activating PPARα. PEA may be a potential treatment for retinopathies with pathological neovascularization and fibrosis.


Assuntos
Agonistas de Receptores de Canabinoides/uso terapêutico , Etanolaminas/uso terapêutico , Gliose/tratamento farmacológico , PPAR alfa/metabolismo , Ácidos Palmíticos/uso terapêutico , Retina/patologia , Neovascularização Retiniana/tratamento farmacológico , Administração Oral , Animais , Western Blotting , Linhagem Celular , Modelos Animais de Doenças , Células Ependimogliais/efeitos dos fármacos , Fibrose/tratamento farmacológico , Fibrose/metabolismo , Fibrose/patologia , Proteína Glial Fibrilar Ácida/genética , Proteína Glial Fibrilar Ácida/metabolismo , Gliose/metabolismo , Gliose/patologia , Marcação In Situ das Extremidades Cortadas , Injeções Intraperitoneais , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oxigênio/toxicidade , PPAR alfa/genética , Ratos , Reação em Cadeia da Polimerase em Tempo Real , Receptores de LDL/genética , Retina/metabolismo , Neovascularização Retiniana/induzido quimicamente , Neovascularização Retiniana/metabolismo , Neovascularização Retiniana/patologia
8.
PLoS One ; 15(3): e0229769, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32150581

RESUMO

Cerebral ischemia/reperfusion (I/R) injury causes cognitive deficits, excitotoxicity, neuroinflammation, oxidative stress and brain edema. Vitamin K2 (Menaquinone 4, MK-4) as a potent antioxidant can be a good candidate to ameliorate I/R consequences. This study focused on the neuroprotective effects of MK-4 for cerebral I/R insult in rat's hippocampus. The rat model of cerebral I/R was generated by transient bilateral common carotid artery occlusion for 20 min. Rats were divided into control, I/R, I/R+DMSO (solvent (1% v/v)) and I/R+MK-4 treated (400 mg/kg, i.p.) groups. Twenty-four hours after I/R injury induction, total brain water content, superoxide dismutase (SOD) activity, nitrate/nitrite concentration and neuronal density were evaluated. In addition to quantify the apoptosis processes, TUNEL staining, as well as expression level of Bax and Bcl2, were assessed. To evaluate astrogliosis and induced neurotoxicity by I/R GFAP and GLT-1 mRNA expression level were quantified. Furthermore, pro-inflammatory cytokines including IL-1ß, IL-6 and TNF-α were measured. Seven days post I/R, behavioral analysis to quantify cognitive function, as well as Nissl staining for surviving neuronal evaluation, were conducted. The findings indicated that administration of MK-4 following I/R injury improved anxiety-like behavior, short term and spatial learning and memory impairment induced by I/R. Also, MK-4 was able to diminish the increased total brain water content, apoptotic cell density, Bax/ Bcl2 ratio and GFAP mRNA expression following I/R. In addition, the high level of nitrate/nitrite, IL-6, IL-1ß and TNF-α induced by I/R was reduced after MK-4 administration. However, MK-4 promotes the level of SOD activity and GLT-1 mRNA expression in I/R rat model. The findings demonstrated that MK-4 can rescue transient global cerebral I/R consequences via its anti-inflammatory and anti-oxidative stress features. MK-4 administration ameliorates neuroinflammation, neurotoxicity and neuronal cell death processes and leads to neuroprotection.


Assuntos
Isquemia Encefálica/tratamento farmacológico , Fármacos Neuroprotetores/uso terapêutico , Traumatismo por Reperfusão/tratamento farmacológico , Vitamina K 2/análogos & derivados , Animais , Apoptose , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Transportador 2 de Aminoácido Excitatório/genética , Transportador 2 de Aminoácido Excitatório/metabolismo , Proteína Glial Fibrilar Ácida/genética , Proteína Glial Fibrilar Ácida/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Masculino , Fármacos Neuroprotetores/farmacologia , Ratos , Ratos Wistar , Aprendizagem Espacial , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Vitamina K 2/farmacologia , Vitamina K 2/uso terapêutico
9.
Arterioscler Thromb Vasc Biol ; 40(5): 1231-1238, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32160776

RESUMO

OBJECTIVE: While GFAP (glial fibrillary acidic protein) is commonly used as a classical marker for astrocytes in the central nervous system, GFAP-expressing progenitor cells give rise to other cell types during development. The goal of this study was to investigate whether GFAP-expressing progenitor cells contribute to the development of vascular cells in major arteries. Approach and Results: To label GFAP-expressing progenitor cells and their progeny, we crossed GFAP promoter-driven Cre recombinase mice (GFAP-Cre) with transgenic mice expressing the Cre-dependent mTmG dual fluorescent reporter gene. Using this genetic fate-mapping approach, here we demonstrate that GFAP-positive progenitor cells contribute to the development of vascular smooth muscle cells in both neural crest- and non-neural crest-derived vascular beds. In addition, GFAP-positive progenitor cells contribute to a subset of endothelial cells in some vasculature. Furthermore, fate-mapping analyses at multiple time points of mouse development demonstrate a time-dependent increase in the contribution of GFAP-positive progenitors to vascular smooth muscle cells, which mostly occurs in the postnatal period. CONCLUSIONS: Our study demonstrates that vascular smooth muscle cells and endothelial cells within the same vascular segment are developmentally heterogeneous, where varying proportions of vascular smooth muscle cells and endothelial cells are contributed by GFAP-positive progenitor cells.


Assuntos
Diferenciação Celular , Linhagem da Célula , Células Progenitoras Endoteliais/metabolismo , Proteína Glial Fibrilar Ácida/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Crista Neural/metabolismo , Animais , Feminino , Genes Reporter , Proteína Glial Fibrilar Ácida/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Músculo Liso Vascular/embriologia , Crista Neural/embriologia , Fenótipo
10.
Int J Nanomedicine ; 15: 1421-1435, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32184596

RESUMO

Purpose: In this study, we aim to explore the effects of graphene oxide (GO), a derivative of graphene, nanoparticles of four different sizes on the cellular fate of mouse neural stem cells (mNSCs). Methods: GO NPs were characterized with transmission electron microscopy (TEM), scanning electron micrography (SEM), atomic force microscopy (AFM) and Raman Spectra analysis. The cytotoxic effects of the GO NPs of different sizes on the mNSCs were determined using CCK-8 assay, Annexin V-APC/ 7-AAD staining and EdU staining assays. We investigated the biological and the mechanisms of GO NPs on cells using immunofluorescence analysis and quantitative real-time PCR (qPCR). Results: The average hydrodynamic sizes of the GO NPs were 417 nm, 663 nm, 1047 nm, and 4651 nm, with a thickness of approximately 22.5 nm, 17.7 nm, 22.4 nm, and 13.4 nm, respectively. GO NPs of all sizes showed low cytotoxicity at a concentration of 20 µg/mL on the mNSCs. Immunostaining demonstrated that treatment with GO NPs, especially the 663 nm ones, enhanced the self-renewal ability of mNSCs in the absence of EGF and bFGF. Under differentiation medium conditions that are free of mitogenic factors, all the GO NPs, particularly the 4651 nm ones, increased the expression level of Tuj1 and GFAP. With regards to the migration ability, we found that 417 nm GO-NP-treated mNSCs migrated over a longer distance than the control group obviously. In addition, higher expression of Rap1, Vinculin and Paxillin was observed in the GO NP-treated groups compared to the control group. mRNA-Sequence analysis and Western blotting results suggested that the 4651 nm GO NPs triggered positive neuronal differentiation through phosphorylation of ERK1/2 by the downregulating of TRPC2. Conclusion: GO NPs play an important role in the applications of inducing self-renewal and differentiation of mNSC, and are promising in the future for further studies.


Assuntos
Grafite/farmacologia , Nanopartículas/química , Células-Tronco Neurais/efeitos dos fármacos , Animais , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Regulação para Baixo/efeitos dos fármacos , Fator de Crescimento Epidérmico/farmacologia , Proteína Glial Fibrilar Ácida/metabolismo , Grafite/química , Camundongos , Microscopia de Força Atômica , Microscopia Eletrônica de Transmissão , Células-Tronco Neurais/fisiologia , Tamanho da Partícula , Fosforilação/efeitos dos fármacos , Análise Espectral Raman , Canais de Cátion TRPC/metabolismo , Tubulina (Proteína)/metabolismo
11.
PLoS One ; 15(2): e0228222, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32074109

RESUMO

Genetically modified swine disease models are becoming increasingly important for studying molecular, physiological and pathological characteristics of human disorders. Given the limited history of these model systems, there remains a great need for proven molecular reagents in swine tissue. Here, to provide a resource for neurological models of disease, we validated antibodies by immunohistochemistry for use in examining central nervous system (CNS) markers in a recently developed miniswine model of neurofibromatosis type 1 (NF1). NF1 is an autosomal dominant tumor predisposition disorder stemming from mutations in NF1, a gene that encodes the Ras-GTPase activating protein neurofibromin. Patients classically present with benign neurofibromas throughout their bodies and can also present with neurological associated symptoms such as chronic pain, cognitive impairment, and behavioral abnormalities. As validated antibodies for immunohistochemistry applications are particularly difficult to find for swine models of neurological disease, we present immunostaining validation of antibodies implicated in glial inflammation (CD68), oligodendrocyte development (NG2, O4 and Olig2), and neuron differentiation and neurotransmission (doublecortin, GAD67, and tyrosine hydroxylase) by examining cellular localization and brain region specificity. Additionally, we confirm the utility of anti-GFAP, anti-Iba1, and anti-MBP antibodies, previously validated in swine, by testing their immunoreactivity across multiple brain regions in mutant NF1 samples. These immunostaining protocols for CNS markers provide a useful resource to the scientific community, furthering the utility of genetically modified miniswine for translational and clinical applications.


Assuntos
Biomarcadores/metabolismo , Neurofibromatose 1/patologia , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Linhagem da Célula , Modelos Animais de Doenças , Proteína Glial Fibrilar Ácida/metabolismo , Proteínas dos Microfilamentos/metabolismo , Microglia/citologia , Microglia/metabolismo , Neurofibromatose 1/metabolismo , Neurofibromina 1/genética , Fator de Transcrição 2 de Oligodendrócitos/metabolismo , Oligodendroglia/citologia , Oligodendroglia/metabolismo , Suínos
12.
Cell Prolif ; 53(2): e12757, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31916327

RESUMO

OBJECTIVES: To testify that endothelial cells (ECs) induce astrocyte maturation by leukaemia inhibitory factor (LIF) secretion. MATERIALS AND METHODS: In vivo experiments, mice bearing floxed alleles of YAP were crossed with mice expressing a Cre recombinase driven by the endothelial Tek promoter (Tek-Cre) to finally obtain the following three genotypes: YAPf/f , Tek-Cre; YAPf/w , Tek-Cre; and YAPf/f . Retinal vascularization and astrocyte network were evaluated by whole-mount fluorescence and Western blotting. In vitro, experiments were performed in an astrocyte and human microvascular endothelial cell (HMEC-1) coculture model to analyse the mechanisms underlying the effect of endothelial YAP on astrocytes. RESULTS: In vivo, YAPf/f ;Tek-Cre mice showed delayed angiogenesis, sparse vessels and decreased glial fibrillary acidic protein (GFAP)+ astrocytes but aberrant growth of endothelial networks and immature astrocytes (platelet-derived growth factor A, PDGFRA+ astrocytes) overgrowth. In vitro, Yap deletion attenuated the LIF release that delayed the maturation of retinal astrocyte which was consistent with the results of HMEC-1-astrocyte coculture. The effect of YAP overexpression on LIF-LIFR axis in HMEC-1 interferes the GFAP expression of astrocyte. In contrast, LIF protein rescues the astrocytic GFAP expression when EC YAP was inhibited by siRNAs. CONCLUSIONS: We show that EC yes-associated protein (YAP) is not only a critical coactivator of Hippo signalling in retinal vessel development but also plays an essential role in retinal astrocyte maturation by regulating LIF production.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Astrócitos/metabolismo , Fator Inibidor de Leucemia/metabolismo , Retina/metabolismo , Vasos Retinianos/metabolismo , Fatores de Transcrição/metabolismo , Animais , Astrócitos/fisiologia , Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Técnicas de Cocultura/métodos , Células Endoteliais/metabolismo , Células Endoteliais/fisiologia , Feminino , Proteína Glial Fibrilar Ácida/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Neovascularização Fisiológica/fisiologia , Neurogênese/fisiologia , Retina/fisiologia , Vasos Retinianos/fisiologia
13.
Int J Mol Sci ; 21(1)2020 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-31947996

RESUMO

Astrocytes play a major role in the pathogenesis of a range of neurodegenerative diseases, including Alzheimer's disease (AD), undergoing dramatic morphological and molecular changes that can cause potentially both beneficial and detrimental effects. They comprise a heterogeneous population, requiring a panel of specific phenotype markers to identify astrocyte subtypes, changes in function and their relation to pathology. This study aimed to characterise expression of the astrocyte marker N-myc downstream regulated gene 2 (NDRG2) in the ageing brain, investigate the relationship between NDRG2 and a panel of astrocyte markers, and relate NDRG2 expression to pathology. NDRG2 specifically immunolabelled the cell body and radiating processes of astrocytes in the temporal cortex of the Cognitive Function and Ageing Study (CFAS) neuropathology cohort. Expression of NDRG2 did not correlate with other astrocyte markers, including glial fibrillary acidic protein (GFAP), excitatory amino acid transporter 2 (EAAT2) and glutamine synthetase (GS). NDRG2 showed a relationship to AT8+ neurofibrillary tangles (p = 0.001) and CD68+ microglia (p = 0.047), but not ß-amyloid plaques or astrocyte nuclear γH2AX immunoreactivity, a marker of DNA damage response. These findings provide new insight into the astrocyte response to pathology in the ageing brain, and suggest NDRG2 may be a potential target to modulate this response.


Assuntos
Envelhecimento , Doença de Alzheimer/patologia , Encéfalo/metabolismo , Microglia/metabolismo , Emaranhados Neurofibrilares/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/metabolismo , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Astrócitos/citologia , Astrócitos/metabolismo , Encéfalo/patologia , Dano ao DNA , Transportador 2 de Aminoácido Excitatório/metabolismo , Regulação da Expressão Gênica , Proteína Glial Fibrilar Ácida/metabolismo , Glutamato-Amônia Ligase/metabolismo , Humanos , Microglia/patologia , Proteínas Supressoras de Tumor/genética , Proteínas tau/metabolismo
14.
Environ Toxicol ; 35(2): 242-253, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31710167

RESUMO

Fluoride is an environmental contaminant that is ubiquitously present in air, water, and soil. It is commonly added in minute quantity to drinking water, toothpaste, and mouth rinses to prevent tooth decay. Epidemiological findings have demonstrated that exposure to fluoride induced neurodevelopmental toxicity, developmental neurotoxicity, and motor disorders. The neuroprotective effect of clofibrate, a peroxisome proliferator-activated receptor alpha agonist, was investigated in the present study. Forty male Wistar rats were used for this study and randomly grouped into 10 rats per group as control, sodium fluoride (NaF) alone (300 ppm), NaF plus clofibrate (250 mg/kg), and NaF plus lisinopril (10 mg/kg), respectively, for 7 days. NaF was administered in drinking water while clofibrate and lisinopril were administered by oral gavage. Markers of neuronal inflammation and oxidative stress, acetylcholinesterase activity, and neurobehavioral (hanging wire and open field) tests were performed. Immunohistochemistry was performed on brain tissues, and they were probed with glial fibrillary acidic protein, ionized calcium-binding adaptor molecule 1, and cerebellar Ca2+ -binding protein calbindin-D28k. The results showed that NaF significantly increased of oxidative stress and neuroinflammation and inhibited AChE activity. Immunostaining showed reactive astrocytes, microgliosis, loss of dendritic spines, and arborization in Purkinje cells in rats administered only NaF. Neurobehavioral results showed that cotreatment of NaF with clofibrate improved muscular strength and locomotion, reduced anxiety, and significantly reduced astrocytic count. Overall, cotreatment of NaF with either clofibrate or lisinopril showed neuroprotective effects by mitigating neuronal inflammation and oxidative and motor incoordination. Hence, clofibrate could be seen as a novel drug candidate against neurodegeneration and motor disorders.


Assuntos
Ataxia/prevenção & controle , Calbindinas/antagonistas & inibidores , Proteínas de Ligação ao Cálcio/metabolismo , Clofibrato/farmacologia , Proteína Glial Fibrilar Ácida/metabolismo , Proteínas dos Microfilamentos/metabolismo , Fármacos Neuroprotetores/farmacologia , Estresse Oxidativo/efeitos dos fármacos , PPAR alfa/agonistas , Fluoreto de Sódio/toxicidade , Animais , Ataxia/imunologia , Biomarcadores/metabolismo , Fluoretos/farmacologia , Inflamação , Masculino , Distribuição Aleatória , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos
15.
Ann Anat ; 227: 151428, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31610254

RESUMO

Aging is a normal process associated with neurodegenerative changes resulting in decline of cognitive and motor functions. Oxidative stress plays an important role. Controlled ozone (O3) therapy has been proved to induce oxidative preconditioning thus reversing oxidative stress. To the best of our knowledge, this research is the first attempt to investigate whether the antioxidant properties of O3 can ameliorate age-associated structural alterations of the cerebral cortex. Ozone administration (at a dose of 0.7mg/kg intraperitonially, three times a week for eight weeks) produced significant downregulation of tissue malondialdehyde (MDA) and upregulation of glutathione, superoxide dismutase (SOD) and catalase (CAT) within the frontal cortex of aged rats. Sections of the frontal cortex from adult and aged rats were stained with hematoxylin and eosin and analyzed using light microscopy. In addition, quantitative immunohistochemical assessments of the expression of inducible nitric oxide synthase (iNOS), caspase-3, glial fibrillary acidic protein (GFAP), Ki67 and acetylcholinesterase (AChE) were performed. Our results revealed the beneficial effect of O3 in improving the neurodegenerative changes of the cerebral cortex of aged rats. Moreover, this study clarified that O3 exerted its effects via reducing oxidative stress, apoptosis, gliosis as well as improving neurogenesis and cholinergic plasticity. This work added to the previously proved aging - associated neurodegenerative effects and provided a new insight into the promising role of O3 to ameliorate these effects.


Assuntos
Envelhecimento/patologia , Lobo Frontal/patologia , Ozônio/uso terapêutico , Animais , Caspase 3/metabolismo , Catalase/análise , Regulação para Baixo , Lobo Frontal/química , Lobo Frontal/enzimologia , Proteína Glial Fibrilar Ácida/metabolismo , Glutationa/análise , Imuno-Histoquímica , Antígeno Ki-67/metabolismo , Masculino , Malondialdeído/análise , Óxido Nítrico Sintase Tipo II/metabolismo , Ozônio/metabolismo , Ratos , Superóxido Dismutase/análise , Regulação para Cima
16.
Int J Mol Sci ; 20(24)2019 Dec 13.
Artigo em Inglês | MEDLINE | ID: mdl-31847143

RESUMO

Dietary supplementation with omega-3 and omega-6 fatty acids offer cardioprotection against air pollution, but these protections have not been established in the brain. We tested whether diets rich in omega-3 or -6 fatty acids offered neuroprotective benefits, by measuring mitochondrial complex enzyme I, II and IV activities and oxidative stress measures in the frontal cortex, cerebellum, hypothalamus, and hippocampus of male rats that were fed either a normal diet, or a diet enriched with fish oil olive oil, or coconut oil followed by exposure to either filtered air or ozone (0.8 ppm) for 4 h/day for 2 days. Results show that mitochondrial complex I enzyme activity was significantly decreased in the cerebellum, hypothalamus and hippocampus by diets. Complex II enzyme activity was significantly lower in frontal cortex and cerebellum of rats maintained on all test diets. Complex IV enzyme activity was significantly lower in the frontal cortex, hypothalamus and hippocampus of animals maintained on fish oil. Ozone exposure decreased complex I and II activity in the cerebellum of rats maintained on the normal diet, an effect blocked by diet treatments. While diet and ozone have no apparent influence on endogenous reactive oxygen species production, they do affect antioxidant levels in the brain. Fish oil was the only diet that ozone exposure did not alter. Microglial morphology and GFAP immunoreactivity were assessed across diet groups; results indicated that fish oil consistently decreased reactive microglia in the hypothalamus and hippocampus. These results indicate that acute ozone exposure alters mitochondrial bioenergetics in brain and co-treatment with omega-6 and omega-3 fatty acids alleviate some adverse effects within the brain.


Assuntos
Encéfalo/metabolismo , Óleo de Coco/farmacologia , Metabolismo Energético/efeitos dos fármacos , Óleos de Peixe/farmacologia , Mitocôndrias/metabolismo , Azeite de Oliva/farmacologia , Animais , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Ácidos Graxos Ômega-3/farmacologia , Ácidos Graxos Ômega-6/farmacologia , Proteína Glial Fibrilar Ácida/metabolismo , Masculino , Microglia/metabolismo , Ratos , Ratos Endogâmicos WKY
17.
BMC Gastroenterol ; 19(1): 221, 2019 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-31856738

RESUMO

BACKGROUND: Nerve growth factor (NGF) and enteric glial cells (EGCs) are associated with visceral hypersensitivity and gastrointestinal motility disorder, which may represent the pathogenesis of functional dyspepsia (FD). This study aimed to investigate the expression of NGF, its high affinity receptor tropomyosin receptor kinase A (TrkA) and the EGC activation marker glial fibrillary acidic protein (GFAP) in the gastric mucosa of patients with FD and the association of these proteins with dyspeptic symptoms. METHODS: Gastric mucosal biopsies taken from 27 FD patients (9 epigastric pain syndrome (EPS) patients, 7 postprandial distress syndrome (PDS) patients and 11 EPS overlap PDS patients) and 26 control subjects were used for analysis. The expression of NGF, TrkA and GFAP was examined, and the association of these proteins with dyspeptic symptoms, including epigastric pain, postprandial fullness, early satiation and epigastric burning, was analysed. RESULTS: The expression levels of NGF, TrkA, and GFAP in the gastric mucosa were significantly higher in the EPS group, the PDS group, and the EPS overlap PDS group than in the healthy control group. There was no significant difference between the FD subgroups. TrkA colocalized with GFAP, which indicated that TrkA was localized to EGCs, and the expression of TrkA in EGCs was significantly higher in the FD group than in the control group. Changes in the expression of NGF, TrkA, and GFAP were positively correlated with epigastric pain, postprandial fullness and early satiation but had no significant relationship with epigastric burning. CONCLUSIONS: The increased expression of gastric NGF, TrkA and GFAP might be involved in FD pathophysiology and symptom perception.


Assuntos
Dispepsia/metabolismo , Mucosa Gástrica/metabolismo , Proteína Glial Fibrilar Ácida/metabolismo , Fator de Crescimento Neural/metabolismo , Receptor trkA/metabolismo , Dor Abdominal/metabolismo , Adulto , Estudos de Casos e Controles , Dispepsia/etiologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade
18.
PLoS One ; 14(11): e0224633, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31693684

RESUMO

Blood levels of Glial Fibrillary Acidic protein (GFAP) reflect processes associated with different types of CNS injury. Evidence suggests that GFAP is cleaved by caspases during CNS injury, hence positioning GFAP fragments as potential biomarkers of injury-associated processes. We set out to develop an assay detecting the neo-epitope generated by caspase-6 cleavage of GFAP (GFAP-C6), and to assess the ability of GFAP-C6 to reflect pathological processes in patients suffering a cardiac arrest and subsequent global cerebral ischemia. Anti-GFAP-C6 antibodies recognized their specific target sequence, and dilution and spike recoveries in serum were within limits of ±20% reflecting high precision and accuracy of measurements. Intra- and inter-assay CVs were below limits of 10% and 15%, respectively. Serological levels of GFAP-C6 were significantly elevated 72 hours after CA (Mean±SD) (20.39±10.59 ng/mL) compared to time of admission (17.79±10.77 ng/mL, p<0.0001), 24 hours (17.40±7.99 ng/mL, p<0.0001) and 48 hours (17.87±8.56 ng/mL, p<0.0001) after CA, but were not related to neurological outcome at day 180. GFAP-C6 levels at admission, 24, 48, and 72 hours after cardiac arrest correlated with two proteolytic fragments of tau, tau-A (r = 0.30, r = 0.40, r = 0.50, r = 0.53, p < 0.0001) and tau-C (r = 54, r = 0.48, r = 0.55, r = 0.54, p < 0.0001), respectively. GFAP-C6 levels did not correlate with other markers of CNS damage; total tau, NSE and S100B. In conclusion, we developed the first assay detecting a caspase-6 cleaved fragment of GFAP in blood. Increased levels at 72 hours after cardiac arrest as well as moderate correlations between GFAP-C6 and two other blood biomarkers of neurodegeneration suggest the ability of GFAP-C6 to reflect pathological processes of the injured brain. Investigations into the potential of GFAP-C6 in other types of CNS injury are warranted.


Assuntos
Encefalopatias/diagnóstico , Proteína Glial Fibrilar Ácida/sangue , Parada Cardíaca/complicações , Doenças Neurodegenerativas/diagnóstico , Adulto , Biomarcadores/sangue , Encéfalo/patologia , Encefalopatias/sangue , Encefalopatias/etiologia , Encefalopatias/patologia , Caspase 6/metabolismo , Feminino , Proteína Glial Fibrilar Ácida/metabolismo , Parada Cardíaca/sangue , Humanos , Masculino , Doenças Neurodegenerativas/sangue , Doenças Neurodegenerativas/etiologia , Doenças Neurodegenerativas/patologia , Valor Preditivo dos Testes , Estudos Prospectivos , Proteólise , Fatores de Tempo
19.
J Comp Pathol ; 172: 37-47, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31690413

RESUMO

Clinical and experimental studies have demonstrated the neurotoxic and behavioural effects of cadmium. However, the exact pathophysiological mechanism(s) of cadmium neurotoxicity on the human central nervous system (CNS) is not completely understood. A rat blood-brain barrier (BBB) endothelial marker, the endothelial barrier antigen (EBA), has been identified and we have shown previously that an anti-EBA IgG1 antibody exclusively recognizes barrier-competent microvessels in the rat CNS and peripheral nervous system (PNS). Endothelial cells of peripheral tissues or brain regions possessing fenestrated microvascular endothelia do not display immunoreactivity for EBA. Here, we describe the application of sequential indirect immunofluorescence with anti-EBA, and an antibody directed against glial fibrillary acidic protein (GFAP), to evaluate the immunoreactivity patterns and morphological alterations in BBB microvessels and astrocytes, following a single, high dose of cadmium in normal, term-delivered young rats. We detected a moderate reduction in immunoreactivity and number of microvessels labelled by the anti-EBA in the forebrain, cerebellum and midbrain in cadmium-exposed rats compared with normal controls. We observed weakly GFAP-reactive astrocytes displaying cell bodies with ill-defined borders and blurred cytoplasm within the white and grey matter of cadmium-exposed brains. The astrocyte nuclei were markedly enlarged, intensely hyperchromatic and exhibited chromatin condensation with nuclear fragmentation. This study indicates for the first time that EBA is involved in, and could serve as a potentially useful marker for studying, cadmium neurotoxicity in the rat model system.


Assuntos
Antígenos de Superfície/metabolismo , Barreira Hematoencefálica/efeitos dos fármacos , Cádmio/toxicidade , Sistema Nervoso Central/efeitos dos fármacos , Animais , Antígenos de Superfície/efeitos dos fármacos , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Astrócitos/patologia , Barreira Hematoencefálica/patologia , Sistema Nervoso Central/crescimento & desenvolvimento , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Proteína Glial Fibrilar Ácida/efeitos dos fármacos , Proteína Glial Fibrilar Ácida/metabolismo , Imuno-Histoquímica , Masculino , Ratos
20.
Adv Mater ; 31(52): e1905577, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31736175

RESUMO

Hierarchically assembled nanomaterials can find a variety of applications in medicine, energy, and electronics. Here, an automatically controlled dip-pulling method is developed and optimized to generate an unprecedented ordered nano-to-micro hierarchical nanoridge-in-microridge (NiM) structure from a bacteria-specific human-safe virus, the filamentous phage with or without genetically displaying a foreign peptide. The NiM structure is pictured as a window blind with each lath (the microridge) made of parallel phage bundles (the nanoridges). It is independent of the substrate materials supporting it. Surprisingly, it can induce the bidirectional differentiation of stem cells into neurons and astrocytes within a short timeframe (only 8 d) not seen before, which is highly desired because both neurons and astrocytes are needed simultaneously in treating neurodegenerative diseases. Since phages can direct tissue regeneration, template materials formation, sense molecules, and build electrodes, the NiM structures displaying different peptides and on varying materials hold promise in many technologically important fields.


Assuntos
Bacteriófago M13/metabolismo , Nanoestruturas/química , Astrócitos/citologia , Astrócitos/metabolismo , Bacteriófago M13/química , Bacteriófago M13/genética , Diferenciação Celular , Linhagem Celular , Proteína Glial Fibrilar Ácida/genética , Proteína Glial Fibrilar Ácida/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Microscopia de Força Atômica , Nestina/genética , Nestina/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Polilisina/química
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