Влияние трансфузии и гипоксии на клетки модели нейроваскулярной единицы in vitro

Up to 57% of patients develop postoperative delirium after surgery for congenital heart defects (CHD). To reduce cerebral damage in pediatric patients during CHD surgery it is important to find out what inflicts the worse damage: would it be a systemic inflammatory response (SIR) triggered by transfusion, or hypoxia developed in non-transfused patients? In vitro evaluation of hypoxia and SIR effects on the neurovascular unit (NVU) cells might contribute to finding the answer.The aim of the study was to compare the effect of varying severity hypoxia and SIR on the functional activity of NUV cells in vitro.Materials and methods. An in vitro NVU model was designed including neurons, astrocytes and endotheliocytes. The effect of hypoxia on NVU was evaluated in the control (C) and 4 study groups (H 1-4), formed based on O2 content in the medium. The C group NVU were cultivated in standard conditions: N2-75%, O2-20%, CO2-5%; H1: N2-99%, O2-1%; H2: N2- 98%, O2-2%; H3: N2-97%, O2-3 %; H4: N2-96%, O2-4%. The significance of the differences was 0.0125. The effect of interleukin-6 (IL-6) content on NVU was measured by adding to culture medium pediatric patients’ serum with known minimal or maximal SIRS-response. The assessment was made in the Control - an intact NVU model, and 2 study groups – “Minimum” and “Maximum”, i.e. samples with minimum or maximum IL-6 content in culture, respectively. The significance of the differences was 0.017. The cells were incubated at a normothermia regimen for 30 minutes. Then, the functional activity of NVU cells was evaluated by measuring transendothelial resistance (TER) for 24 hours and Lucifer Yellow (LY) permeability test at 60 and 90 minutes after the start of the experiment.Results. After incubation under hypoxic conditions, TER changes occurred in all studied groups. However, they were statistically significant only in the group with 1% oxygen content in the medium. TER decrease in this group was observed after 2, 4 and 24 hours. LY permeability also changed at 60 and 90 minutes, similarly - in NVU cultivated with 1% oxygen in the medium. Minimal TER values were documented at 4 hours after patients’ serum was added to NVU cells culture medium, and TER increased at 24 hours in both study groups. Cellular permeability to LY changed significantly after 1 hour exposure in both groups - with minimum and maximum IL-6 content in the medium. Although at 90 minutes, there was no difference between the 3 groups in LY permeability tests.Conclusion: Intensive SIR demonstrated short-term but more deleterious than hypoxia, effect on cells in the NVU model. Hypoxia disrupted functional activity of NUV cells only at 1% O 2 concentration in the medium.Частота развития послеоперационного делирия при коррекции врожденных пороков сердца (ВПС) достигает 57%. В поиске путей профилактики церебрального повреждения при коррекции ВПС у детей важным является вопрос - что опаснее: гипоксия при отказе от трансфузии или действие повышенного системного воспалительного ответа (СВО) при ее применении. Исследование действия гипоксии и СВО на клетки нейроваскулярной единицы (НВЕ) in vitro способствует решению данного вопроса.Цель исследования: сравнить влияние гипоксии различной выраженности и системного воспалительного ответа на функциональную активность клеток нейроваскулярной единицы.Материалы и методы. Сформировали in vitro модель НВЕ, состоящую из нейронов, астроцитов и эндотелиоцитов. Влияние гипоксии на НВЕ оценивали в контрольной (К) и 4 исследуемых (Г1-4) группах. Группы сформировали по содержанию О2 в среде: К – стандартные условия культивирования: N2-75%, O2-20%, CO2-5%; Г1: N2-99 %, O2-1 %; Г2: N2-98 %, O2-2 %; Г3: N2-97 %, O2-3 %; Г4: N2-96 %, O2-4 %. Значимость различий составила 0,0125. Влияние содержания интерлейкина-6 (ИЛ-6) на НВЕ определяли при культивировании клеток с добавлением сыворотки крови пациентов детского возраста с минимальным, либо максимальным напряжением СВО. Оценку провели в контрольной и 2 исследуемых группах: «Контроль» – интактная модель НВЕ; группы «Минимум» и «Максимум» - образцы с минимальным либо максимальным содержанием ИЛ-6 в культуре соответственно. Значимость различий составила 0,017. Инкубацию клеток проводили в режиме нормотермии в течение 30 минут. Затем оценивали функциональную активность клеток НВЕ методом измерения трансэндотелиального сопротивления (ТЭС) в течение 24 часов и измерения проницаемости для красителя Lucifer Yellow (LY) через 60 и 90 минут от начала эксперимента.Результаты. После инкубации в условиях гипоксии изменения ТЭС наступили во всех исследуемых группах клеток. Однако, только в группе с 1% содержанием кислорода в среде они были статистически значимы. Снижения ТЭС в данной группе наблюдали через 2, 4 и 24 часа. Проницаемость клеток для красителя LY изменилась через 60 и 90 минут также только в условиях их инкубации в среде с 1 % кислородом. При культивировании клеток НВЕ с сывороткой крови пациентов выявили минимальные значения ТЭС через 4 часа и их дальнейшее повышение через 24 часа для обеих исследуемых групп НВЕ. Проницаемость клеток для LY значительно изменилась к 60-й минуте как в группе с минимальным, так и с максимальным содержанием ИЛ-6 в среде. При этом к 90-й минуте различий этого показателя в исследуемых группах и в контрольной группе уже не наблюдали.Заключение. Напряженный СВО оказал более выраженное, но кратковременное действие на модель НВЕ, чем гипоксия. Гипоксия нарушила функциональную активность НВЕ только при концентрации кислорода в среде - 1 %

Early transcriptome changes associated with western diet induced NASH in Ldlr−/− mice points to activation of hepatic macrophages and an acute phase response

BackgroundNonalcoholic fatty liver disease (NAFLD) is a global health problem. Identifying early gene indicators contributing to the onset and progression of NAFLD has the potential to develop novel targets for early therapeutic intervention. We report on the early and late transcriptomic signatures of western diet (WD)-induced nonalcoholic steatohepatitis (NASH) in female and male Ldlr−/− mice, with time-points at 1 week and 40 weeks on the WD. Control Ldlr−/− mice were maintained on a low-fat diet (LFD) for 1 and 40 weeks.MethodsThe approach included quantitation of anthropometric and hepatic histology markers of disease as well as the hepatic transcriptome.ResultsOnly mice fed the WD for 40 weeks revealed evidence of NASH, i.e., hepatic steatosis and fibrosis. RNASeq transcriptome analysis, however, revealed multiple cell-specific changes in gene expression after 1 week that persisted to 40 weeks on the WD. These early markers of disease include induction of acute phase response (Saa1-2, Orm2), fibrosis (Col1A1, Col1A2, TGFβ) and NASH associated macrophage (NAM, i.e., Trem2 high, Mmp12 low). We also noted the induction of transcripts associated with metabolic syndrome, including Mmp12, Trem2, Gpnmb, Lgals3 and Lpl. Finally, 1 week of WD feeding was sufficient to significantly induce TNFα, a cytokine involved in both hepatic and systemic inflammation.ConclusionThis study revealed early onset changes in the hepatic transcriptome that develop well before any anthropometric or histological evidence of NALFD or NASH and pointed to cell-specific targeting for the prevention of disease progression

Designing in vitro Blood-Brain Barrier Models Reproducing Alterations in Brain Aging

Blood-brain barrier (BBB) modeling in vitro is a huge area of research covering study of intercellular communications and development of BBB, establishment of specific properties that provide controlled permeability of the barrier. Current approaches in designing new BBB models include development of new (bio) scaffolds supporting barriergenesis/angiogenesis and BBB integrity; use of methods enabling modulation of BBB permeability; application of modern analytical techniques for screening the transfer of metabolites, bio-macromolecules, selected drug candidates and drug delivery systems; establishment of 3D models; application of microfluidic technologies; reconstruction of microphysiological systems with the barrier constituents. Acceptance of idea that BBB in vitro models should resemble real functional activity of the barrier in different periods of ontogenesis and in different (patho) physiological conditions leads to proposal that establishment of BBB in vitro model with alterations specific for aging brain is one of current challenges in neurosciences and bioengineering. Vascular dysfunction in the aging brain often associates with leaky BBB, alterations in perivascular microenvironment, neuroinflammation, perturbed neuronal and astroglial activity within the neurovascular unit, impairments in neurogenic niches where microvascular scaffold plays a key regulatory role. The review article is focused on aging-related alterations in BBB and current approaches to development of “aging” BBB models in vitro

Differential Roles of Environmental Enrichment in Alzheimer’s Type of Neurodegeneration and Physiological Aging

Impairment of hippocampal adult neurogenesis in aging or degenerating brain is a well-known phenomenon caused by the shortage of brain stem cell pool, alterations in the local microenvironment within the neurogenic niches, or deregulation of stem cell development. Environmental enrichment (EE) has been proposed as a potent tool to restore brain functions, to prevent aging-associated neurodegeneration, and to cure neuronal deficits seen in neurodevelopmental and neurodegenerative disorders. Here, we report our data on the effects of environmental enrichment on hippocampal neurogenesis in vivo and neurosphere-forming capacity of hippocampal stem/progenitor cells in vitro. Two models – Alzheimer’s type of neurodegeneration and physiological brain aging – were chosen for the comparative analysis of EE effects. We found that environmental enrichment greatly affects the expression of markers specific for stem cells, progenitor cells and differentiated neurons (Pax6, Ngn2, NeuroD1, NeuN) in the hippocampus of young adult rats or rats with Alzheimer’s disease (AD) model but less efficiently in aged animals. Application of time-lag mathematical model for the analysis of impedance traces obtained in real-time monitoring of cell proliferation in vitro revealed that EE could restore neurosphere-forming capacity of hippocampal stem/progenitor cells more efficiently in young adult animals (fourfold greater in the control group comparing to the AD model group) but not in the aged rats (no positive effect of environmental enrichment at all). In accordance with the results obtained in vivo, EE was almost ineffective in the recovery of hippocampal neurogenic reserve in vitro in aged, but not in amyloid-treated or young adult, rats. Therefore, EE-based neuroprotective strategies effective in Aβ-affected brain could not be directly extrapolated to aged brain

Plasticity of Adipose Tissue-Derived Stem Cells and Regulation of Angiogenesis

Adipose tissue is recognized as an important organ with metabolic, regulatory, and plastic roles. Adipose tissue-derived stem cells (ASCs) with self-renewal properties localize in the stromal vascular fraction (SVF) being present in a vascular niche, thereby, contributing to local regulation of angiogenesis and vessel remodeling. In the past decades, ASCs have attracted much attention from biologists and bioengineers, particularly, because of their multilineage differentiation potential, strong proliferation, and migration abilities in vitro and high resistance to oxidative stress and senescence. Current data suggest that the SVF serves as an important source of endothelial progenitors, endothelial cells, and pericytes, thereby, contributing to vessel remodeling and growth. In addition, ASCs demonstrate intriguing metabolic and interlineage plasticity, which makes them good candidates for creating regenerative therapeutic protocols, in vitro tissue models and microphysiological systems, and tissue-on-chip devices for diagnostic and regeneration-supporting purposes. This review covers recent achievements in understanding the metabolic activity within the SVF niches (lactate and NAD+ metabolism), which is critical for maintaining the pool of ASCs, and discloses their pro-angiogenic potential, particularly, in the complex therapy of cardiovascular and cerebrovascular diseases

Abstracts from the 20th International Symposium on Signal Transduction at the Blood-Brain Barriers

https://deepblue.lib.umich.edu/bitstream/2027.42/138963/1/12987_2017_Article_71.pd

Molecular mechanisms of proteins - targets for SARS-CoV-2 (review)

The rapidly accumulating information about the new coronavirus infection and the ambiguous results obtained by various authors necessitate further research aiming at prevention and treatment of this disease. At the moment, there is convincing evidence that the pathogen affects not only the respiratory but also the central nervous system (CNS). The aim of the study is to provide an insight into the molecular mechanisms underlying the damage to the CNS caused by the new coronavirus SARS-CoV-2. Results. By analyzing the literature, we provide evidence that the brain is targeted by this virus. SARS-CoV-2 enters the body with the help of the target proteins: angiotensin-converting enzyme 2 (ACE2) and associated serine protease TMPRSS2 of the nasal epithelium. Brain damage develops before the onset of pulmonary symptoms. The virus spreads through the brain tissue into the piriform cortex, basal ganglia, midbrain, and hypothalamus. Later, the substantia nigra of the midbrain, amygdala, hippocampus, and cerebellum become affected. Massive death of neurons, astrogliosis and activation of microglia develop at the next stage of the disease. By day 4, an excessive production of proinflammatory cytokines in the brain, local neuroinflammation, breakdown of the blood-brain barrier, and impaired neuroplasticity are detected. These changes imply the involvement of a vascular component driven by excessive activity of matrix metalloproteinases, mediated by CD147. The main players in the pathogenesis of COVID-19 in the brain are products of angiotensin II (AT II) metabolism, largely angiotensin 1-7 (AT 1-7) and angiotensin IV (AT IV). There are conflicting data regarding their role in damage to the CNS in various diseases, including the coronavirus infection. The second participant in the pathogenesis of brain damage in COVID-19 is CD147 - the inducer of extracellular matrix metalloproteinases. This molecule is expressed on the endothelial cells of cerebral microvessels, as well as on leukocytes present in the brain during neuroinflammation. The CD147 molecule plays a significant role in maintaining the structural and functional integrity of the blood-brain barrier by controlling the basal membrane permeability and by mediating the astrocyte-endothelial interactions. Via the above mechanisms, an exposure to SARS-CoV-2 leads to direct damage to the neurovascular unit of the brain

Neuroinflammation and Infection: Molecular Mechanisms Associated with Dysfunction of Neurovascular Unit

Neuroinflammation is a complex inflammatory process in the central nervous system, which is sought to play an important defensive role against various pathogens, toxins or factors that induce neurodegeneration. The onset of neurodegenerative diseases and various microbial infections are counted as stimuli that can challenge the host immune system and trigger the development of neuroinflammation. The homeostatic nature of neuroinflammation is essential to maintain the neuroplasticity. Neuroinflammation is regulated by the activity of neuronal, glial, and endothelial cells within the neurovascular unit, which serves as a “platform” for the coordinated action of pro- and anti-inflammatory mechanisms. Production of inflammatory mediators (cytokines, chemokines, reactive oxygen species) by brain resident cells or cells migrating from the peripheral blood, results in the impairment of blood-brain barrier integrity, thereby further affecting the course of local inflammation. In this review, we analyzed the most recent data on the central nervous system inflammation and focused on major mechanisms of neurovascular unit dysfunction caused by neuroinflammation and infections