Human In Vitro Models of Neuroenergetics and Neurometabolic Disturbances: Current Advances and Clinical Perspectives.

Neurological conditions conquer the world; they are the leading cause of disability and the second leading cause of death worldwide, and they appear all around the world in every age group, gender, nationality, and socioeconomic class. Despite the growing evidence of an immense impact of perturbations in neuroenergetics on overall brain function, only little is known about the underlying mechanisms. Especially human insights are sparse, owing to a shortage of physiologically relevant model systems. With this perspective, we aim to explore the key steps and considerations involved in developing an advanced human in vitro model for studying neuroenergetics. We discuss biological and technological strategies to meet the requirements of a predictive model, aiming at providing a guide and inspiration for future in vitro models of neuroenergetics.

Protocol for designing and bioprinting multi-layered constructs to reconstruct an endothelial-epithelial 3D model.

3D bioprinting has opened new possibilities and elevated tissue engineering complexity. Here, we present a protocol to design a 3D model with two cell lineage layers (A549 and HUVEC) to recreate multi-cell constructs. We describe the steps for slicing the constructs, handling hydrogels, and detailing the bioprinting setup. These 3D-bioprinted constructs can be adapted to various cell models-from primary cell cultures to commercial cell lines and induced pluripotent stem cells (IPCs)-and applications, including drug screening and disease modeling. For complete details on the use and execution of this protocol, please refer to Cruz et al.1.

A Gelatin Methacrylate-Based Hydrogel as a Potential Bioink for 3D Bioprinting and Neuronal Differentiation.

Neuronal loss is the ultimate pathophysiologic event in central nervous system (CNS) diseases and replacing these neurons is one of the most significant challenges in regenerative medicine. Providing a suitable microenvironment for new neuron engraftment, proliferation, and synapse formation is a primary goal for 3D bioprinting. Among the various biomaterials, gelatin methacrylate (GelMA) stands out due to its Arg-Gly-Asp (RGD) domains, which assure its biocompatibility and degradation under physiological conditions. This work aimed to produce different GelMA-based bioink compositions, verify their mechanical and biological properties, and evaluate their ability to support neurogenesis. We evaluated four different GelMA-based bioink compositions; however, when it came to their biological properties, incorporating extracellular matrix components, such as GeltrexTM, was essential to ensure human neuroprogenitor cell viability. Finally, GeltrexTM: 8% GelMA (1:1) bioink efficiently maintained human neuroprogenitor cell stemness and supported neuronal differentiation. Interestingly, this bioink composition provides a suitable environment for murine astrocytes to de-differentiate into neural stem cells and give rise to MAP2-positive cells.

Overexpression of mir-135b and mir-210 in mesenchymal stromal cells for the enrichment of extracellular vesicles with angiogenic factors.

Extracellular vesicles (EVs) are known as molecular carriers involved in cell communication and the regulation of (patho)physiological processes. miRNAs and growth factors are the main contents of EVs which make them a good candidate for the treatment of diseases caused by ischemia, but the low production of EVs by a cell producer and a significant variation of the molecular contents in EVs according to the cell source are the main limitations of their widespread use. Here, we show how to improve the therapeutic properties of mesenchymal stromal cell (MSC)-derived EVs (MSC-EVs) by modifying MSCs to enrich these EVs with specific angiomiRs (miR-135b or miR-210) using lentiviral vectors carrying miR-135b or miR-210. MSCs were obtained from the mouse bone marrow and transduced with a corresponding lentivector to overexpress miR-135b or miR-210. The EVs were then isolated by ultracentrifugation and characterized using a flow cytometer and a nanoparticle tracking analyzer. The levels of 20 genes in the MSCs and 12 microRNAs in both MSCs and EVs were assessed by RT‒qPCR. The proangiogenic activity of EVs was subsequently assessed in human umbilical vein endothelial cells (HUVECs). The results confirmed the overexpression of the respective microRNA in modified MSCs. Moreover, miR-135b overexpression upregulated miR-210-5p and follistatin, whereas the overexpression of miR-210 downregulated miR-221 and upregulated miR-296. The tube formation assay showed that EVs from MSCs overexpressing miR-210-5p (EVmiR210) significantly promoted tubular structure formation in HUVECs. A significant increase in angiogenic proteins (PGF, endothelin 1, and artemin) and genes (VEGF, activin A, and IGFBP1) in HUVECs treated with VEmiR210 justifies the better tubular structure formation of these cells compared with that of EVmiR135b-treated HUVECs, which showed upregulated expression of only artemin. Collectively, our results show that the EV cargo can be modified by lentiviral vectors to enrich specific miRNAs to achieve a specific angiogenic potential.

A new function for Prokineticin 2: Recruitment of SVZ-derived neuroblasts to the injured cortex in a mouse model of traumatic brain injury.

Traumatic brain injury is an important cause of global morbidity and mortality. After an initial injury, there is a cascade of cellular and molecular events that ultimately lead to cell death. Therapies aim to both counteract these mechanisms and replenish the lost cell population in order to improve recovery. The adult mammal brain has at least two neurogenic regions that maintain physiological functions: the subgranular zone of the dentate gyrus in the hippocampus, which produces neurons that integrate locally, and the subventricular zone (SVZ) adjacent to the lateral ventricles, which produces neuroblasts that migrate through the rostral migratory stream (RMS) to the olfactory bulbs. Brain injuries, as well as neurodegenerative diseases, induce the SVZ to respond by increasing cell proliferation and migration to the injured areas. Here we report that cells migrate from the SVZ and RMS to the injured cortex after traumatic brain injury in mice, and that the physiological RMS migration is not impaired. We also show that Prokineticin 2 (PROK2), a chemokine important for the olfactory bulb neurogenesis, expressed exclusively by cortical microglia in the cortex as early as 24 h after injury. We then show that administration of a PROK2 receptor antagonist decreases the number of SVZ cells that reach the injured cortex, while injection of recombinant PROK2 into the cortex of uninjured mice attracts SVZ cells. We also demonstrate that cells expressing PROK2 in vitro directionally attract SVZ cells. These data suggest that PROK2 could be utilized in regeneration efforts for the acutely injured mammalian cortex.

Right-to-left shunt and the hypercoagulable state: does paradoxical embolism play a role in patients with antiphospholipid syndrome and stroke?

OBJECTIVE: Patent foramen ovale is associated with paradoxical embolism (PE) and stroke. Hypercoagulable states, such as antiphospholipid syndrome (APS), can exacerbate PE by increasing clot formation. The aim of this study was to verify whether patients with APS and stroke present a right-to-left shunt (RLS) with greater frequency than patients with APS but without stroke. METHODS: Fifty-three patients with APS were tested for RLS using contrast-enhanced transcranial Doppler (cTCD): 23 patients had a history of stroke (Stroke Group) and 30 had no history of stroke (No-stroke Group). RESULTS: cTCD was positive in 15 patients (65%) from the Stroke Group and in 16 patients (53%) in the No-stroke Group (p=0.56). The proportion of patients with a small RLS (<10 high-intensity transient sign or HITS) and a large RLS (>10 HITS) was similar between the groups without significant difference. CONCLUSIONS: Our data do not support the theory that paradoxical embolism may play an important role in stroke in APS patients.

Right-to-left shunt and the hypercoagulable state: does paradoxical embolism play a role in patients with antiphospholipid syndrome and stroke? / Shunt direita-esquerda e hipercoagulabilidade: embolia paradoxal pode ser responsável pelo AVC na síndrome antifosfolipídica?

OBJECTIVE: Patent foramen ovale is associated with paradoxical embolism (PE) and stroke. Hypercoagulable states, such as antiphospholipid syndrome (APS), can exacerbate PE by increasing clot formation. The aim of this study was to verify whether patients with APS and stroke present a right-to-left shunt (RLS) with greater frequency than patients with APS but without stroke. METHODS: Fifty-three patients with APS were tested for RLS using contrast-enhanced transcranial Doppler (cTCD): 23 patients had a history of stroke (Stroke Group) and 30 had no history of stroke (No-stroke Group). RESULTS: cTCD was positive in 15 patients (65%) from the Stroke Group and in 16 patients (53%) in the No-stroke Group (p=0.56). The proportion of patients with a small RLS (<10 high-intensity transient sign or HITS) and a large RLS (>10 HITS) was similar between the groups without significant difference. CONCLUSIONS: Our data do not support the theory that paradoxical embolism may play an important role in stroke in APS patients.

OBJETIVO: O forame oval patente está associado com embolia paradoxal e acidente vascular cerebral isquêmico (AVCi). Estados de hipercoagulabilidade, como a síndrome antifosfolipídica (SAF), podem facilitar esse processo, aumentando a formação de coágulos. O objetivo deste estudo foi verificar se pacientes com SAF e AVCi apresentam maior frequência de shunt direita-esquerda (SDE), comparados a pacientes com SAF sem AVCi. MÉTODOS: Cinquenta e três pacientes com SAF foram testados para SDE usando Doppler transcraniano contrastado (DTCc): 23 com AVCi (Grupo AVC) e 30 sem história de AVCi (Grupo Controle). RESULTADOS: DTCc foi positivo em 15 pacientes (65%) do Grupo AVC e em 16 pacientes (53%) no Grupo Controle (p=0,56). A proporção de pacientes com pequeno SDE (<10 HITS) e grande SDE (>10 HITS) foi semelhante nos dois grupos. Não houve diferença significativa entre os grupos. CONCLUSÕES: Nossos dados não sugerem que embolia paradoxal seja causa importante de AVCi em pacientes com SAF.