An Improved Method for Physical Separation of Cerebral Vasculature and Parenchyma Enables Detection of Blood-Brain-Barrier Dysfunction

The neurovascular niche is crucial for constant blood supply and blood-brain barrier (BBB) function and is altered in a number of different neurological conditions, making this an intensely active field of research. Brain vasculature is unique for its tight association of endothelial cells with astrocytic endfeet processes. Separation of the vascular compartment by centrifugation-based methods confirmed enrichment of astrocytic endfeet processes, making it possible to study the entire vascular niche with such methods. Several centrifugation-based separation protocols are found in the literature; however, with some constraints which limit their applicability and the scope of the studies. Here, we describe and validate a protocol for physically separating the neurovascular niche from the parenchyma, which is optimized for smaller tissue quantities. Using endothelial, neuronal, and astrocyte markers, we show that quantitative Western blot-based target detection can be performed of both the vessel-enriched and parenchymal fractions using as little as a single mouse brain hemisphere. Validation of our protocol in rodent stroke models by detecting changes in tight junction protein expression, serum albumin signals and astrocyte activation, i.e., increased glial fibrillary acidic protein expression, between the ipsilateral and the lesion-free contralateral hemisphere demonstrates this protocol as a new way of detecting BBB breakdown and astrogliosis, respectively.

Embryonic periventricular endothelial cells demonstrate a unique pro-neurodevelopment and anti-inflammatory gene signature

Brain embryonic periventricular endothelial cells (PVEC) crosstalk with neural progenitor cells (NPC) promoting mutual proliferation, formation of tubular-like structures in the former and maintenance of stemness in the latter. To better characterize this interaction, we conducted a comparative transcriptome analysis of mouse PVEC vs. adult brain endothelial cells (ABEC) in mono-culture or NPC co-culture. We identified > 6000 differentially expressed genes (DEG), regardless of culture condition. PVEC exhibited a 30-fold greater response to NPC than ABEC (411 vs. 13 DEG). Gene Ontology (GO) analysis of DEG that were higher or lower in PVEC vs. ABEC identified “Nervous system development” and “Response to Stress” as the top significantly different biological process, respectively. Enrichment in canonical pathways included HIF1A, FGF/stemness, WNT signaling, interferon signaling and complement. Solute carriers (SLC) and ABC transporters represented an important subset of DEG, underscoring PVEC’s implication in blood–brain barrier formation and maintenance of nutrient-rich/non-toxic environment. Our work characterizes the gene signature of PVEC and their important partnership with NPC, underpinning their unique role in maintaining a healthy neurovascular niche, and in supporting brain development. This information may pave the way for additional studies to explore their therapeutic potential in neuro-degenerative diseases, such as Alzheimer’s and Parkinson’s disease.

An Improved Method for Physical Separation of Cerebral Vasculature and Parenchyma Enables Detection of Blood-Brain-Barrier Dysfunction

The neurovascular niche is crucial for constant blood supply and blood-brain barrier (BBB) function and is altered in a number of different neurological conditions, making this an intensely active field of research. Brain vasculature is unique for its tight association of endothelial cells with astrocytic endfeet processes. Separation of the vascular compartment by centrifugation-based methods confirmed enrichment of astrocytic endfeet processes, making it possible to study the entire vascular niche with such methods. Several centrifugation-based separation protocols are found in the literature; however, with some constraints which limit their applicability and the scope of the studies. Here, we describe and validate a protocol for physically separating the neurovascular niche from the parenchyma, which is optimized for smaller tissue quantities. Using endothelial, neuronal and astrocyte markers, we show that quantitative Western blot-based target detection can be performed of both the vascular and parenchymal fractions using as little as a single mouse brain hemisphere. Validation of our protocol in rodent stroke models by detecting changes in serum albumin signals and astrocyte activation, i.e. increased glial fibrillary acidic protein expression, between the ipsilateral and the lesion-free contralateral hemisphere demonstrates this protocol as a new way of detecting BBB breakdown and astrogliosis, respectively.

Enhanced angiogenesis by the hyaluronic acid hydrogels immobilized with a VEGF mimetic peptide in a traumatic brain injury model in rats

Angiogenesis plays an important role in brain injury repair, which contributes to the reconstruction of regenerative neurovascular niche for promoting axonal regeneration in the lesion area. As a major component of developing brain extracellular matrix, hyaluronic acid (HA) has attracted more attention as a supporting matrix for brain repair. In the present study, HA-KLT hydrogel was developed via modifying HA with a VEGF mimetic peptide of KLT (KLTWQELYQLKYKGI). The characterization of the hydrogel shows that it could provide a porous, three-dimensional scaffold structure, which has a large specific surface area available for cell adhesion and interaction. Compared with the unmodified HA hydrogel, the HA-KLT hydrogel could effectively promote the attachment, spreading and proliferation of endothelial cells in vitro. Furthermore, the pro-angiogenic ability of hydrogels in vivo was evaluated by implanting them into the lesion cavities in the injured rat brain. Our results showed that the hydrogels could form a permissive interface with the host tissues at 4 weeks after implantation. Moreover, they could efficiently inhibit the formation of glial scars at the injured sites. The HA-KLT hydrogel could significantly increase the expression of endoglin/CD105 and promote the formation of blood vessels, suggesting that HA-KLT hydrogel promoted angiogenesis in vivo. Collectively, the HA-KLT hydrogel has the potential to repair brain defects by promoting angiogenesis and inhibiting the formation of glial-derived scar tissue.