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1.
Microcirculation ; 21(6): 478-89, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24495210

RESUMO

OBJECTIVE: To isolate, purify, and cultivate primary retinal microvascular pericytes (RMPs) from rats to facilitate the study of their properties in vitro. METHODS: Primary RMPs were isolated from weanling rats by mechanical morcel and collagenase digestion, and purified by a step-wise combination of selective medium with different glucose concentrations, medium exchange, and partial enzymatic digestion. Morphology of RMPs was assessed by phase contrast microscopy. Further characterization was analyzed by immunofluorescence. Functional assay was evaluated by the pericytes- endothelial cells (ECs) coculture system. RESULTS: Retinal microvascular pericytes migrated out of microvascular fragments after 24-48 hours of plating and reached subconfluence on days 14-16. The cells showed typical pericyte morphology with large irregular triangular cell bodies and multiple long processes, and uniformly expressed the cellular markers α-SMA, PDGFR-ß, NG2 and desmin, but were negative for vWF, GS, GFAP and SMMHC. Ninety-nine percent of the cell population had double positive staining for α-SMA and PDGFR-ß. In the coculture system, RMPs can directly contact ECs and move together to form the capillary-like cords. CONCLUSIONS: Retinal microvascular pericytes can be readily obtained by our method. We report the first cultivation of primary RMPs from rats and establish a simple method for their isolation and purification.


Assuntos
Antígenos de Diferenciação/metabolismo , Separação Celular , Pericitos , Retina , Animais , Técnicas de Cocultura , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Masculino , Pericitos/citologia , Pericitos/metabolismo , Ratos , Ratos Sprague-Dawley , Retina/citologia , Retina/metabolismo
2.
Int Immunopharmacol ; 132: 112030, 2024 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-38603861

RESUMO

Mast cells (MCs) play a significant role in various diseases, and their activation and degranulation can trigger inflammatory responses and barrier damage. Several studies have indicated that vagus nerve stimulation (VNS) exerts ameliorates neurological injury, and regulates gut MC degranulation. However, there is limited research on the modulatory effect of VNS on MCs in both the gut and brain in brain ischemia-reperfusion (I/R) injury in this process. We aim to develop a minimally invasive, targeted and convenient VNS approach to assess the impact of VNS and to clarify the relationship between VNS and MCs on the prognosis of acute ischemic stroke. We utilized middle cerebral artery occlusion/reperfusion (MCAO/r) to induce brain I/R injury. After the experiment, the motor function and neurofunctional impairments of the rats were detected, and the gastrointestinal function, blood-brain barrier (BBB) and intestinal barrier damage, and systemic and local inflammation were evaluated by Nissl, TTC staining, Evans blue, immunofluorescence staining, transmission electron microscopy, western blot assays, ELISA, and fecal 16S rRNA sequencing methods. Our research confirmed that our minimally invasive VNS method is a novel approach for stimulating the vagus nerve. VNS alleviated motor deficits and gastrointestinal dysfunction while also suppressing intestinal and neuroinflammation. Additionally, VNS ameliorated gut microbiota dysbiosis in rats. Furthermore, our analysis indicated that VNS reduces chymase secretion by modulating MCs degranulation and improves intestinal and BBB damage. Our results showed that VNS treatment can alleviate the damage of BBB and colonic barrier after cerebral I/R by modulating mast cell degranulation, and alleviates systemic inflammatory responses.


Assuntos
Barreira Hematoencefálica , Eixo Encéfalo-Intestino , Degranulação Celular , Microbioma Gastrointestinal , AVC Isquêmico , Mastócitos , Ratos Sprague-Dawley , Traumatismo por Reperfusão , Estimulação do Nervo Vago , Animais , Mastócitos/imunologia , Estimulação do Nervo Vago/métodos , Masculino , Ratos , Traumatismo por Reperfusão/terapia , Traumatismo por Reperfusão/imunologia , AVC Isquêmico/terapia , Eixo Encéfalo-Intestino/fisiologia , Infarto da Artéria Cerebral Média/terapia , Modelos Animais de Doenças , Isquemia Encefálica/terapia , Isquemia Encefálica/imunologia
3.
Dis Markers ; 2021: 7124835, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34630739

RESUMO

Inner blood-retina barrier (iBRB) is primarily formed of retinal microvascular endothelial cells (ECs) with tight junctions, which are surrounded and supported by retinal microvascular pericytes (RMPs) and basement membrane. Pericytes are believed to be critically involved in the physiology and pathology of iBRB. However, the underlying mechanism remains to be fully elucidated. We developed a novel in vitro iBRB model which was composed of primary cultures of rat retinal ECs and RMPs based on Transwell system. We tested the involvement of pericytes in the migration and invasion of ECs, examined the expression and activity of matrix metalloproteinase- (MMP-) 2/MMP-9 in the culture, evaluated the TEER and permeability of iBRB, and assessed the expression of ZO-1, occludin, claudin-5, and VE-cadherin of endothelial junctions. We found that RMPs with indirect contact of ECs can increase the expression of MMP-2 and upgrade the activity of MMP-2/9 in the coculture, which subsequently decreased TJ protein abundance of ZO-1 and occludin in ECs, promoted the migration of ECs, and finally reduced the integrity of iBRB. Taken together, our data show that RMP relative location with ECs is involved in the integrity of iBRB via MMP-2/9 and has important implications for treating diabetic retinopathy and other retinal disorders involving iBRB dysfunction.


Assuntos
Barreira Hematorretiniana/citologia , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Pericitos/citologia , Retina/citologia , Animais , Barreira Hematorretiniana/metabolismo , Movimento Celular , Células Cultivadas , Técnicas de Cocultura , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Masculino , Modelos Biológicos , Pericitos/metabolismo , Cultura Primária de Células , Ratos , Retina/metabolismo , Junções Íntimas/metabolismo
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