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Abstract The vasculature of the brain consists of specialized endothelial cells that form a blood-brain barrier (BBB). This barrier, in conjunction with supporting cell types, forms the neurovascular unit (NVU). The NVU restricts the passage of certain substances from the bloodstream while selectively permitting essential nutrients and molecules to enter the brain. This protective role is crucial for optimal brain function, but presents a significant obstacle in treating neurological conditions, necessitating chemical modifications or advanced drug delivery methods for most drugs to cross the NVU. A deeper understanding of NVU in health and disease will aid in the identification of new therapeutic targets and drug delivery strategies for improved treatment of neurological disorders. To achieve this goal, we need models that reflect the human BBB and NVU in health and disease. Although animal models of the brain’s vasculature have proven valuable, they are often of limited translational relevance due to interspecies differences or inability to faithfully mimic human disease conditions. For this reason, human in vitro models are essential to improve our understanding of the brain’s vasculature under healthy and diseased conditions. This review delves into the advancements in in vitro modeling of the BBB and NVU, with a particular focus on microfluidic models. After providing a historical overview of the field, we shift our focus to recent developments, offering insights into the latest achievements and their associated constraints. We briefly examine the importance of chip materials and methods to facilitate fluid flow, emphasizing their critical roles in achieving the necessary throughput for the integration of microfluidic models into routine experimentation. Subsequently, we highlight the recent strides made in enhancing the biological complexity of microfluidic NVU models and propose recommendations for elevating the biological relevance of future iterations. Importantly, the NVU is an intricate structure and it is improbable that any model will fully encompass all its aspects. Fit-for-purpose models offer a valuable compromise between physiological relevance and ease-of-use and hold the future of NVU modeling: as simple as possible, as complex as needed.

Abstract The neurovascular unit (NVU) is a highly organized multicellular system localized in the brain, formed by neuronal, glial (astrocytes, oligodendrocytes, and microglia) and vascular (endothelial cells and pericytes) cells. The blood–brain barrier, a complex and dynamic endothelial cell barrier in the brain microvasculature that separates the blood from the brain parenchyma, is a component of the NVU. In a variety of neurological disorders, including Alzheimer’s disease, multiple sclerosis, and stroke, dysfunctions of the NVU occurs. There is, however, a lack of knowledge regarding the NVU function in leukodystrophies, which are rare monogenic disorders that primarily affect the white matter. Since leukodystrophies are rare diseases, human brain tissue availability is scarce and representative animal models that significantly recapitulate the disease are difficult to develop. The introduction of human induced pluripotent stem cells (hiPSC) now makes it possible to surpass these limitations while maintaining the ability to work in a biologically relevant human context and safeguarding the genetic background of the patient. This review aims to provide further insights into the NVU functioning in leukodystrophies, with a special focus on iPSC-derived models that can be used to dissect neurovascular pathophysiology in these diseases.

Abstract Background In ischemic stroke, the function of the cerebral vasculature is impaired. This vascular structure is formed by the so-called neurovascular unit (NVU). A better understanding of the mechanisms involved in NVU dysfunction and recovery may lead to new insights for the development of highly sought therapeutic approaches. To date, there remains an unmet need for complex human in vitro models of the NVU to study ischemic events seen in the human brain. Methods We here describe the development of a human NVU on-a-chip model using a platform that allows culture of 40 chips in parallel. The model comprises a perfused vessel of primary human brain endothelial cells in co-culture with induced pluripotent stem cell derived astrocytes and neurons. Ischemic stroke was mimicked using a threefold approach that combines chemical hypoxia, hypoglycemia, and halted perfusion. Results Immunofluorescent staining confirmed expression of endothelial adherens and tight junction proteins, as well as astrocytic and neuronal markers. In addition, the model expresses relevant brain endothelial transporters and shows spontaneous neuronal firing. The NVU on-a-chip model demonstrates tight barrier function, evidenced by retention of small molecule sodium fluorescein in its lumen. Exposure to the toxic compound staurosporine disrupted the endothelial barrier, causing reduced transepithelial electrical resistance and increased permeability to sodium fluorescein. Under stroke mimicking conditions, brain endothelial cells showed strongly reduced barrier function (35-fold higher apparent permeability) and 7.3-fold decreased mitochondrial potential. Furthermore, levels of adenosine triphosphate were significantly reduced on both the blood- and the brain side of the model (4.8-fold and 11.7-fold reduction, respectively). Conclusions The NVU on-a-chip model presented here can be used for fundamental studies of NVU function in stroke and other neurological diseases and for investigation of potential restorative therapies to fight neurological disorders. Due to the platform’s relatively high throughput and compatibility with automation, the model holds potential for drug compound screening.

Abstract Background RNA sequencing (RNA-Seq) in its varied forms has become an indispensable tool for analyzing differential gene expression and thus characterization of specific tissues. Aiming to understand the brain barriers genetic signature, RNA seq has also been introduced in brain barriers research. This has led to availability of both, bulk and single-cell RNA-Seq datasets over the last few years. If appropriately performed, the RNA-Seq studies provide powerful datasets that allow for significant deepening of knowledge on the molecular mechanisms that establish the brain barriers. However, RNA-Seq studies comprise complex workflows that require to consider many options and variables before, during and after the proper sequencing process. Main body In the current manuscript, we build on the interdisciplinary experience of the European PhD Training Network BtRAIN ( https://www.btrain-2020.eu/ ) where bioinformaticians and brain barriers researchers collaborated to analyze and establish RNA-Seq datasets on vertebrate brain barriers. The obstacles BtRAIN has identified in this process have been integrated into the present manuscript. It provides guidelines along the entire workflow of brain barriers RNA-Seq studies starting from the overall experimental design to interpretation of results. Focusing on the vertebrate endothelial blood–brain barrier (BBB) and epithelial blood-cerebrospinal-fluid barrier (BCSFB) of the choroid plexus, we provide a step-by-step description of the workflow, highlighting the decisions to be made at each step of the workflow and explaining the strengths and weaknesses of individual choices made. Finally, we propose recommendations for accurate data interpretation and on the information to be included into a publication to ensure appropriate accessibility of the data and reproducibility of the observations by the scientific community. Conclusion Next generation transcriptomic profiling of the brain barriers provides a novel resource for understanding the development, function and pathology of these barrier cells, which is essential for understanding CNS homeostasis and disease. Continuous advancement and sophistication of RNA-Seq will require interdisciplinary approaches between brain barrier researchers and bioinformaticians as successfully performed in BtRAIN. The present guidelines are built on the BtRAIN interdisciplinary experience and aim to facilitate collaboration of brain barriers researchers with bioinformaticians to advance RNA-Seq study design in the brain barriers community.