Blood Exposure Causes Ventricular Zone Disruption and Glial Activation In Vitro

Abstract Intraventricular hemorrhage (IVH) is the most common cause of pediatric hydrocephalus in North America but remains poorly understood. Cell junction-mediated ventricular zone (VZ) disruption and astrogliosis are associated with the pathogenesis of congenital, nonhemorrhagic hydrocephalus. Recently, our group demonstrated that VZ disruption is also present in preterm infants with IVH. On the basis of this observation, we hypothesized that blood triggers the loss of VZ cell junction integrity and related cytopathology. In order to test this hypothesis, we developed an in vitro model of IVH by applying syngeneic blood to cultured VZ cells obtained from newborn mice. Following blood treatment, cells were assayed for N-cadherin-dependent adherens junctions, ciliated ependymal cells, and markers of glial activation using immunohistochemistry and immunoblotting. After 24–48 hours of exposure to blood, VZ cell junctions were disrupted as determined by a significant reduction in N-cadherin expression (p < 0.05). This was also associated with significant decrease in multiciliated cells and increase in glial fibrillary acid protein-expressing cells (p < 0.05). These observations suggest that, in vitro, blood triggers VZ cell loss and glial activation in a pattern that mirrors the cytopathology of human IVH and supports the relevance of this in vitro model to define injury mechanisms.

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Preterm intraventricular hemorrhage in vitro: modeling the cytopathology of the ventricular zone

10.21203/rs.3.rs-16876/v3 ◽

2020 ◽

Author(s):

Leandro Castaneyra-Ruiz ◽

James P. McAllister ◽

Diego M. Morales ◽

Steven L. Brody ◽

Albert M. Isaacs ◽

...

Keyword(s):

Intraventricular Hemorrhage ◽

In Vitro Model ◽

Ventricular Zone ◽

Ependymal Cells ◽

High Temporal Resolution ◽

Neuroepithelial Cells ◽

Vitro Model ◽

Developmental Features ◽

The Impact

Abstract Background: Severe intraventricular hemorrhage (IVH) is one of the most devastating neurological complications in preterm infants, with the majority suffering long-term neurological morbidity and up to 50 percent developing post-hemorrhagic hydrocephalus (PHH). Despite the importance of this disease, its cytopathological mechanisms are not well known. An in vitro model of IVH is required to investigate the effects of blood and its components on the developing ventricular zone (VZ) and its stem cell niche. To address this need, we developed a protocol from our accepted in vitro model to mimic the cytopathological conditions of IVH in the preterm infant. Methods: Maturing neuroepithelial cells from the VZ were harvested from the entire lateral ventricles of wild type C57BL/6 mice at 1-4 days of age and expanded in proliferation media for 3-5 days. At confluence, cells were re-plated onto 24-well plates in differentiation media to generate ependymal cells (EC). At approximately 3-5 days, which corresponded to the onset of EC differentiation based on the appearance of multiciliated cells, phosphate-buffered saline for controls or syngeneic whole blood for IVH was added to the EC surface. The cells were examined for the expression of EC markers of differentiation and maturation to qualitatively and quantitatively assess the effect of blood exposure on VZ transition from neuroepithelial cells to EC. Discussion: This protocol will allow investigators to test cytopathological mechanisms contributing to the pathology of IVH with high temporal resolution and query the impact of injury to the maturation of the VZ. This technique recapitulates features of normal maturation of the VZ in vitro, offering the capacity to investigate the developmental features of VZ biogenesis.

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Response of Astrocytes to Blood Exposure due to Shunt Insertion in vitro

10.22541/au.162145883.36815443/v1 ◽

2021 ◽

Author(s):

Mira Zaranek ◽

Rooshan Arshad ◽

Kevin Zheng ◽

Carolyn Harris

Keyword(s):

In Vitro Model ◽

Cell Attachment ◽

Ventricular Zone ◽

Total Cell ◽

Total Cell Count ◽

Shunt Insertion ◽

Catheter Obstruction ◽

Vitro Model ◽

Number Of Cells

The breakdown of the ventricular zone (VZ) with the presence of blood in cerebrospinal fluid (CSF) has been shown to increase shunt catheter obstruction in the treatment of hydrocephalus, but the mechanisms by which this occurs are generally unknown. Using a custom-built incubation chamber, we immunofluorescently assayed cell attachment and morphology on shunt catheters with and without blood after 14 days. Samples exposed to blood showed significantly increased cell attachment (average total cell count 392.0±317.1 versus control of 94.7±44.5, P<0.0001). Analysis of the glial fibrillary acidic protein (GFAP) expression showed similar trends (854.4±450.7 versus control of 174.3±116.5, P<0.0001). An in vitro model was developed to represent the exposure of astrocytes to blood following an increase in BBB permeability. Exposure of astrocytes to blood increases the number of cells and their spread on the shunt.

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Coupling primary and stem cell–derived cardiomyocytes in an in vitro model of cardiac cell therapy

The Journal of Cell Biology ◽

10.1083/jcb.201508026 ◽

2016 ◽

Vol 212 (4) ◽

pp. 389-397 ◽

Cited By ~ 26

Author(s):

Yvonne Aratyn-Schaus ◽

Francesco S. Pasqualini ◽

Hongyan Yuan ◽

Megan L. McCain ◽

George J.C. Ye ◽

...

Keyword(s):

Stem Cell ◽

Cell Therapy ◽

In Vitro Model ◽

Cell Junction ◽

Cardiac Cell ◽

Force Transmission ◽

Cardiac Cell Therapy ◽

Vitro Model ◽

Cell Cell

The efficacy of cardiac cell therapy depends on the integration of existing and newly formed cardiomyocytes. Here, we developed a minimal in vitro model of this interface by engineering two cell microtissues (μtissues) containing mouse cardiomyocytes, representing spared myocardium after injury, and cardiomyocytes generated from embryonic and induced pluripotent stem cells, to model newly formed cells. We demonstrated that weaker stem cell–derived myocytes coupled with stronger myocytes to support synchronous contraction, but this arrangement required focal adhesion-like structures near the cell–cell junction that degrade force transmission between cells. Moreover, we developed a computational model of μtissue mechanics to demonstrate that a reduction in isometric tension is sufficient to impair force transmission across the cell–cell boundary. Together, our in vitro and in silico results suggest that mechanotransductive mechanisms may contribute to the modest functional benefits observed in cell-therapy studies by regulating the amount of contractile force effectively transmitted at the junction between newly formed and spared myocytes.

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Preterm Intraventricular Hemorrhage in vitro: Modeling the Cytopathology of the Ventricular Zone

10.21203/rs.3.rs-16876/v2 ◽

2020 ◽

Author(s):

Leandro Castaneyra-Ruiz ◽

James P. McAllister ◽

Diego M. Morales ◽

Steven L. Brody ◽

Albert M. Isaacs ◽

...

Keyword(s):

Intraventricular Hemorrhage ◽

In Vitro Model ◽

Ependymal Cell ◽

Ventricular Zone ◽

High Temporal Resolution ◽

Neuroepithelial Cells ◽

Vitro Model ◽

Developmental Features ◽

The Impact

Abstract Background: Severe intraventricular hemorrhage (IVH) is one of the most devastating neurological complications in preterm infants, with the majority suffering long-term neurological morbidity and up to 50 percent developing post hemorrhagic hydrocephalus (PHH). Despite the importance of this disease, its cytopathological mechanisms are not well known. An in vitro model of IVH is required to investigate the effects of blood and its components on the developing ventricular zone (VZ) and its stem cell niche. To address this need, we developed a new in vitro model to mimic the cytopathological conditions of IVH in the preterm infant. Methods: Maturing neuroepithelial cells from the VZ were harvested from the entire lateral ventricles of wild type C57BL/6 mice at 1-4 days of age and expanded in proliferation media for 3-5 days. At confluence, cells were re-plated onto 24-well plates in differentiation media to generate ependymal cells (EC). At approximately 3-5 days, which corresponded to the onset of ependymal cell differentiation based on the appearance of multiciliated cells , phosphate-buffered saline for controls or syngeneic whole blood for IVH was added to the ependymal cell surface. The cells were examined for the expression of EC markers of differentiation and maturation to qualitatively and quantitatively assess the effect of blood exposure on VZ transition from neuroepithelial cells to EC. Discussion: This model will allow investigators to test cytopathological mechanisms contributing to the pathology of IVH with high temporal resolution and query the impact of injury to the maturation of the VZ. This technique recapitulates features of normal maturation of the VZ in vitro, offering the capacity to investigate the developmental features of VZ biogenesis.

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Preterm Intraventricular Hemorrhage in vitro: Modeling the Cytopathology of the Ventricular Zone

10.21203/rs.3.rs-16876/v1 ◽

2020 ◽

Author(s):

Leandro Castaneyra-Ruiz ◽

James P. McAllister ◽

Diego M. Morales ◽

Steven L. Brody ◽

Albert M. Isaacs ◽

...

Keyword(s):

Intraventricular Hemorrhage ◽

In Vitro Model ◽

Ependymal Cell ◽

Ventricular Zone ◽

High Temporal Resolution ◽

Neuroepithelial Cells ◽

Vitro Model ◽

Developmental Features ◽

The Impact

Abstract Background: Severe intraventricular hemorrhage (IVH) is one of the most devastating neurological complications in preterm infants, with the majority suffering long-term neurological morbidity and up to 50 percent developing post hemorrhagic hydrocephalus (PHH). Despite the importance of this disease, its cytopathological mechanisms are not well known. An in vitro model of IVH is required to investigate the effects of blood and its components on the developing ventricular zone (VZ) and its stem cell niche. To address this need, we developed a new in vitro model to mimic the cytopathological conditions of IVH in the preterm infant. Methods: Maturing neuroepithelial cells from the VZ were harvested from the entire lateral ventricles of wild type C57BL/6 mice at 1-4 days of age and expanded in proliferation media for 3-5 days. At confluence, cells were re-plated onto 24-well plates in differentiation media to generate ependymal cells (EC). At approximately 3-5 days, which corresponded to the onset of ependymal cell differentiation based on the appearance of multiciliated cells , phosphate-buffered saline for controls or syngeneic whole blood for IVH was added to the ependymal cell surface. The cells were examined for the expression of EC markers of differentiation and maturation to qualitatively and quantitatively assess the effect of blood exposure on VZ transition from neuroepithelial cells to EC. Discussion: This model will allow investigators to test cytopathological mechanisms contributing to the pathology of IVH with high temporal resolution and query the impact of injury to the maturation of the VZ. This technique recapitulates features of normal maturation of the VZ in vitro, offering the capacity to investigate the developmental features of VZ biogenesis.

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An in vitro model for investigation of vitamin A effects on wound healing

International Journal for Vitamin and Nutrition Research ◽

10.1024/0300-9831/a000692 ◽

2021 ◽

pp. 1-6

Author(s):

Hoda Keshmiri Neghab ◽

Mohammad Hasan Soheilifar ◽

Gholamreza Esmaeeli Djavid

Keyword(s):

Wound Healing ◽

Endothelial Cell ◽

Vitamin A ◽

In Vitro Model ◽

Cellular Proliferation ◽

Endothelial Cell Migration ◽

Normal Fibroblast ◽

Anti Inflammatory ◽

Vitro Model

Abstract. Wound healing consists of a series of highly orderly overlapping processes characterized by hemostasis, inflammation, proliferation, and remodeling. Prolongation or interruption in each phase can lead to delayed wound healing or a non-healing chronic wound. Vitamin A is a crucial nutrient that is most beneficial for the health of the skin. The present study was undertaken to determine the effect of vitamin A on regeneration, angiogenesis, and inflammation characteristics in an in vitro model system during wound healing. For this purpose, mouse skin normal fibroblast (L929), human umbilical vein endothelial cell (HUVEC), and monocyte/macrophage-like cell line (RAW 264.7) were considered to evaluate proliferation, angiogenesis, and anti-inﬂammatory responses, respectively. Vitamin A (0.1–5 μM) increased cellular proliferation of L929 and HUVEC (p < 0.05). Similarly, it stimulated angiogenesis by promoting endothelial cell migration up to approximately 4 fold and interestingly tube formation up to 8.5 fold (p < 0.01). Furthermore, vitamin A treatment was shown to decrease the level of nitric oxide production in a dose-dependent effect (p < 0.05), exhibiting the anti-inflammatory property of vitamin A in accelerating wound healing. These results may reveal the therapeutic potential of vitamin A in diabetic wound healing by stimulating regeneration, angiogenesis, and anti-inﬂammation responses.

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