Reconstituting 3D Neurovascular Unit In Vitro With Primary Neural Stem Cells And Brain Microvascular Endothelial Cells Of Rats

Background The neurovascular unit(NVU)is an organized multicellular and multicomponent network that is important for brain health.This brain-localized unit is composed of neural and vascular components,and the interface and interactions between these components are crucial for the regulation of material exchange between the bloodstream and the brain parenchyma.The neural component of the NVU consists of neurons and glial cells(microglia,astrocytes,oligodendrocytes),while the vascular component consists of endothelial cells,pericytes,and vascular smooth muscle cells.The neural component—particularly the neurons—are crucial for the cognitive function of patients with various neurodegeneration diseases and are affected by a variety of pathological influences(e.g.,hypoxia,oxidative stress).The vascular component—particularly the endothelial cells—forms the blood–brain barrier(BBB),which is a part of the NVU and a critical multicellular vascular structure that separates the brain from systemic blood circulation.Continuous intercellular junctional protein complexes between adjacent endothelium help to maintain the physical and functional integrity of the BBB,by strictly regulating the permeability through the BBB and transferring extracellular mechanical signals to the surrounding cells.The importance of vascular contributions to various brain disorders,including stroke and Alzheimer’s disease,is becoming increasingly recognized.Neurovascular dysfunction is a primary or secondary cause and central processin the pathogenesis of several cerebrovascular and neurodegenerative disorders,including stroke.Therefore,the overall protection of the neurovascular unit(NVU)is a promising therapeutic strategy for various neurovascular diseases.However,the complexity of the NVU limits the study of the pathological mechanisms of neurovascular dysfunction.Reconstituting the in vitro NVU is an urgent need for the pathological study and drug screening of neurovascular diseases.ObjectivesIn the present study,we aimed at(i)presenting a method for reconstituting the in vitro brain 3D NVU with primary NSCs and brain microvascular endothelial cells(BMECs)of rats in the Matrigel ECM system,and exploring the pathological changes of the 3D NVU in the context of ischemia to evaluate its applicability to pathological study,employing a well-characterized in vitro oxygen–glucose deprivation(OGD)pattern;(ii)investigating the potential NVU protection of VEGF,Edaravone,or catalpol in OGD-exposed 3D NVU or ischemic stroke rats from the aspects of vascular and neuronal protection,angiogenesis and neurogenesis promotion,and oxidative stress.MethodsFirstly,primary neural stem cells and brain microvascular endothelial cells were isolated and cultured form new born rats,followed by the co-culture of the two cell types in Matrigel to generat an in vitro three-dimensional NVU(3D NVU)with primary neural stem cells(NSCs)and brain microvascular endothelial cells(BMECs).Confocal imaging and immunocytochemistry,transmission electron microscopy(TEM),electrophysiological recordings,permeability measurements,and protein expression analysis were used to quantitatively assess the NVU characteristics of structure and function.Additionally,we transplanted the 3D NVU to the infarcted side of cerebral ischemia rats to examine the functional effects of the grafted 3D NVU on the host ischemia brain.Finally,we modeled ischemic stroke-induced pathological NVU by employing widely recognized oxygen-glucose deprivation(OGD)pattern,to investigate the potential NVU protection of VEGF/Edaravone,provides evidences of the applicability in pathological study for 3D NVU.Furthermore,the potential roles of catalpol in NVU protection and its possible underlying mechanisms were explored and dissected,both in ischemic stroke rats and in vitro pathological three-dimensional NVU model induced by oxygen-glucose deprivation,and the protection effects were compared between in vivo and in vitro.ResultsIn this study,we generated a spontaneously assembled three-dimensional NVU(3D NVU)by employing the primary neural stem cells and brain microvascular endothelial cells in a Matrigel extracellular matrix platform.This novel model exhibits the fundamental structures and features of the NVU,including neurons,astrocytes,oligodendrocytes,vascular-like structures,and blood–brain barrier-like characteristics.Additionally,under oxygen-glucose deprivation,the 3D NVU exhibits the neurovascular-or oxidative stress-related pathological characteristics of cerebral ischemia and the injuries can be mitigated respectively by supplementing with the vascular endothelial growth factor or edaravone,which demonstrated that the availability of 3D NVU in ischemic stroke modeling.Finally,the 3D NVU promoted the angiogenesis and neurogenesis in the brain of cerebral ischemia rats.We expect that the proposed in vitro 3D NVU model will be widely used to investigate the relationships between angiogenesis and neurogenesis and to study the pathology and pharmacology of neurovascular diseases.Next,catalpol was used to further demonstrate the 3D NVU was suitable for pharmacological study.We paid close attention to study whether catalpol exhibits protection effects for NVU in ischemic stroke and its underlying mechanisms.The results showed that catalpol reduced neurological deficit scores and infarct volume,protected vascular structures and neuronal morphology,and promoted angiogenesis and neurogenesis in ischemic stroke.Furthermore,both of the in vivo and in vitro results demonstrated that vascular endothelial growth factor(VEGF)was increased by catalpol,followed by the activation of PI3K/AKT and MEK1/2/ERK1/2 pathways.Collectively,we conclude that catalpol promotes VEGF production via activating PI3K/AKT,then the PI3K/AKT and MEK1/2/ERK1/2 pathways signals are enhanced by VEGF,both in ischemic hemisphere and in vitro NVU model,may trigger a feedforward loop to protect NVU from ischemic stroke.The results suggested that catalpol improved impaired NVU in ischemia by protecting vessel-neuron-astrocyte structures and morphology,and promoting angiogenesis and neurogenesis to replenish lost vessels and neurons.Both the in vivo and in vitro results also showed that the pathological changes of OGD-exposed 3D NVU in vitro and the ameliorative effects of catalpol were in good agreement with that in vivo.Conclusions(ⅰ)Primary NSCs and BMECs were co-cultured in a 3D Matrigel matrix,which was a robust method for generating a 3D NVU with functional vascular-like structures,BBB-like characteristics,and diverse nerve cells,including neurons,astrocytes,and oligodendrocytes.This 3D NVU system facilitates the accurate physiological representation of the brain by mimicking the interplay between neural and vascular components as well as the complex in vivo cellular interactions and structures.(ⅱ)After the transplantation of the 3D NVU to the infarcted side of the cerebral ischemia rats,it still had biological activity and could promote the neurogenesis and angiogenesis and reduce the infarcted volume of the ischemic side.These results indicate that the 3D NVU constructed from primary cells is functional and can be used to investigate neurogenesis and angiogenesis in cases of cerebral ischemia.(ⅲ)Additionally,our 3D NVU model can be used to mimic the pathological changes and investigate angiogenesis and neurogenesis in the context of ischemia,using a well-characterized in vitro OGD pattern.The proposed 3D NVU model also provides a valuable platform and precise spatiotemporal control for neurogenesis and angiogenesis research and the investigation of brain functions,which rely on interactions among brain cells,drug screening studies as a therapeutic strategy,and clinical applications(e.g.,in the case of degenerative disease).(ⅳ)Moreover,catalpol protects NVU from ischemic stroke by improving vascular and neuronal morphology,maintaining barrier function,and promoting angiogenesis and neurogenesis,then alleviates neurological deficits,reduces infarction volume of ischemic rats.We proposed that the primary and detailed mechanisms of NVU protection of catalpol is dependent on PI3K/AKT to promote VEGF production and then mediated NVU protection by increasing FAK and Paxillin expression levels,and activating VEGF-PI3K/AKT and VEGF-MEK1/2/ERK1/2 pathways,in a partly feedforward loop manner.The results suggested that the 3D NVU can be used to explore the mechanisms of NVU dysfunction,screen and evaluate the NVU protection drugs in vitro.