Mechanobiological Modulation of In Vitro Astrocyte Reactivity Using Variable Gel Stiffness.

After traumatic brain injury, the brain extracellular matrix undergoes structural rearrangement due to changes in matrix composition, activation of proteases, and deposition of chondroitin sulfate proteoglycans by reactive astrocytes to produce the glial scar. These changes lead to a softening of the tissue, where the stiffness of the contusion "core" and peripheral "pericontusional" regions becomes softer than that of healthy tissue. Pioneering mechanotransduction studies have shown that soft substrates upregulate intermediate filament proteins in reactive astrocytes; however, many other aspects of astrocyte biology remain unclear. Here, we developed a platform for the culture of cortical astrocytes using polyacrylamide (PA) gels of varying stiffness (measured in Pascal; Pa) to mimic injury-related regions in order to investigate the effects of tissue stiffness on astrocyte reactivity and morphology. Our results show that substrate stiffness influences astrocyte phenotype; soft 300 Pa substrates led to increased GFAP immunoreactivity, proliferation, and complexity of processes. Intermediate 800 Pa substrates increased Aggrecan+, Brevican+, and Neurocan+ astrocytes. The stiffest 1 kPa substrates led to astrocytes with basal morphologies, similar to a physiological state. These results advance our understanding of astrocyte mechanotransduction processes and provide evidence of how substrates with engineered stiffness can mimic the injury microenvironment.

High Concentrations of Cannabidiol Induce Neurotoxicity in Neurosphere Culture System.

Recent studies have demonstrated that cannabinoids are potentially effective in the treatment of various neurological conditions, and cannabidiol (CBD), one of the most studied compounds, has been proposed as a non-toxic option. However, the adverse effects of CBD on neurodevelopmental processes have rarely been studied in cell culture systems. To better understand CBD's influence on neurodevelopment, we exposed neural progenitor cells (NPCs) to different concentrations of CBD (1 µM, 5 µM, and 10 µM). We assessed the morphology, migration, differentiation, cell death, and gene expression in 2D and 3D bioprinted models to stimulate physiological conditions more effectively. Our results showed that CBD was more toxic at higher concentrations (5 µM and 10 µM) and affected the viability of NPCs than at lower concentrations (1 µM), in both 2D and 3D models. Moreover, our study revealed that higher concentrations of CBD drastically reduced the size of neurospheres and the number of NPCs within neurospheres, impaired the morphology and mobility of neurons and astrocytes after differentiation, and reduced neurite sprouting. Interestingly, we also found that CBD alters cellular metabolism by influencing the expression of glycolytic and ß-oxidative enzymes in the early and late stages of metabolic pathways. Therefore, our study demonstrated that higher concentrations of CBD promote important changes in cellular functions that are crucial during CNS development.

Therapeutic Potential of Targeting Prokineticin Receptors in Diseases.

The prokineticins (PKs) were discovered approximately 20 years ago as small peptides inducing gut contractility. Today, they are established as angiogenic, anorectic, and proinflammatory cytokines, chemokines, hormones, and neuropeptides involved in variety of physiologic and pathophysiological pathways. Their altered expression or mutations implicated in several diseases make them a potential biomarker. Their G-protein coupled receptors, PKR1 and PKR2, have divergent roles that can be therapeutic target for treatment of cardiovascular, metabolic, and neural diseases as well as pain and cancer. This article reviews and summarizes our current knowledge of PK family functions from development of heart and brain to regulation of homeostasis in health and diseases. Finally, the review summarizes the established roles of the endogenous peptides, synthetic peptides and the selective ligands of PKR1 and PKR2, and nonpeptide orthostatic and allosteric modulator of the receptors in preclinical disease models. The present review emphasizes the ambiguous aspects and gaps in our knowledge of functions of PKR ligands and elucidates future perspectives for PK research. SIGNIFICANCE STATEMENT: This review provides an in-depth view of the prokineticin family and PK receptors that can be active without their endogenous ligand and exhibits "constitutive" activity in diseases. Their non- peptide ligands display promising effects in several preclinical disease models. PKs can be the diagnostic biomarker of several diseases. A thorough understanding of the role of prokineticin family and their receptor types in health and diseases is critical to develop novel therapeutic strategies with safety concerns.

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.

Sleep deprivation modulates APOE and LDL receptor-related protein 1 through thyroid hormone T4 and impairs Aß clearance in hippocampus of rats.

Alzheimer's disease is the most common form of dementia. One of its pathological hallmarks is Aß accumulation, which is influenced by APOE genotype and expression, as well as by sleep homeostasis. However, conflicting mechanisms for APOE roles in Aß clearance have been reported, and the relationship between APOE and sleep also remains unclear. In this study, we aimed to investigate how hormonal alteration caused by sleep deprivation affects APOE and its receptors in rats, and to evaluate the role of different cell types in Aß clearance. Paradoxical sleep deprivation for 96 h increased Aß level in hippocampus with concomitant reduction of APOE and LRP1 at the time point within the resting period. Sleep deprivation also significantly reduced T4 levels in both active and resting times. To evaluate the effect of T4 variation, C6 glial cells and primary brain endothelial cells were treated with T4. High T4 level (300 ng/mL) increased APOE, but reduced LRP1 and LDL-R in C6 cells, while in primary endothelial cells, LDL-R levels were increased. Treatment of C6 cells with exogenous APOE reduced LRP1 and Aß uptake. These results suggest that T4 modulates LRP1 and LDL-R in both cell types, but in the opposite manner, thus, sleep deprivation might modify the ratio of the receptors in blood-brain barrier and glial cells by altering T4 levels. Considering that LRP1 and LDL-R are important for Aß clearance, sleep deprivation might also affect the degree of participation of glia in Aß clearance, and consequently, turnover of Aß in the brain.

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.

Simple and efficient protocol to isolate and culture brain microvascular endothelial cells from newborn mice.

The neurovascular unit (NVU) is a multicellular structure comprising of neurons, glial cells, and non-neural cells, and it is supported by a specialized extracellular matrix, the basal lamina. Astrocytes, brain microvascular endothelial cells (BMECs), pericytes, and smooth muscle cells constitute the blood-brain barrier (BBB). BMECs have a mesodermal origin and invade the nervous system early in neural tube development, forming the BBB anatomical core. BMECs are connected by adherent junction complexes composed of integral membrane and cytoplasmic proteins. In vivo and in vitro studies have shown that, given the proximity and relationship with neural cells, BMECs acquire a unique gene expression profile, proteome, and specific mechanical and physical properties compared to endothelial cells from the general vasculature. BMECs are fundamental in maintaining brain homeostasis by regulating transcellular and paracellular transport of fluids, molecules, and cells. Therefore, it is essential to gain in-depth knowledge of the dynamic cellular structure of the cells in the NVU and their interactions with health and disease. Here we describe a significantly improved and simplified protocol using C57BL/6 newborn mice at postnatal day 1 (PND1) to isolate, purify, and culture BMECs monolayers in two different substrates (glass coverslips and transwell culture inserts). In vitro characterization and validation of the BMEC primary culture monolayers seeded on glass or insert included light microscopy, immunolabeling, and gene expression profile. Transendothelial electrical resistance (TEER) measurement and diffusion test were used as functional assays for adherent junction complexes and integrity and permeability of BMECs monolayers. The protocol presented here for the isolation and culture of BMECs is more straightforward than previously published protocols and yields a high number of purified cells. Finally, we tested BMECs function using the oxygen-glucose deprivation (OGD) model of hypoxia. This protocol may be suitable as a bioscaffold for secondary cell seeding allowing the study and better understanding of the NVU.

Biomimetic Nanovesicles-Sources, Design, Production Methods, and Applications.

Despite all the progress in the field of liposomes and nanoparticles for applications as drug and gene delivery systems, the specific targeting and immune system escape capabilities of these systems are still limited. Biomimetic nanovesicles emerged as a strategy to overcome these and other limitations associated with synthetic carriers, such as short circulation time, cytotoxicity, and difficulty in crossing biological barriers, since many of the desirable abilities of drug delivery systems are innate characteristics of biological vesicles. Thus, the question arises: would biomimetic nanovesicles be responsible for addressing these advances? It is currently known that biomimetic nanovesicles (BNV) can combine the intrinsic advantages of natural materials with the well-known production methods and controllability of synthetic systems. Besides, the development of the biotechnology and nanotechnology fields has provided a better understanding of the functionalities of biological vesicles and the means for the design and production of biomimetic nanovesicles (BNV). Based on this, this work will focus on tracking the main research on biomimetic nanovesicles (BNV) applied as drug and gene delivery systems, and for vaccines applications. In addition, it will describe the different sources of natural vesicles, the technical perspectives on obtaining them, and the possibility of their hybridization with synthetic liposomes.

Self-organization of in vitro neuronal assemblies drives to complex network topology.

Activity-dependent self-organization plays an important role in the formation of specific and stereotyped connectivity patterns in neural circuits. By combining neuronal cultures, and tools with approaches from network neuroscience and information theory, we can study how complex network topology emerges from local neuronal interactions. We constructed effective connectivity networks using a transfer entropy analysis of spike trains recorded from rat embryo dissociated hippocampal neuron cultures between 6 and 35 days in vitro to investigate how the topology evolves during maturation. The methodology for constructing the networks considered the synapse delay and addressed the influence of firing rate and population bursts as well as spurious effects on the inference of connections. We found that the number of links in the networks grew over the course of development, shifting from a segregated to a more integrated architecture. As part of this progression, three significant aspects of complex network topology emerged. In agreement with previous in silico and in vitro studies, a small-world architecture was detected, largely due to strong clustering among neurons. Additionally, the networks developed in a modular topology, with most modules comprising nearby neurons. Finally, highly active neurons acquired topological characteristics that made them important nodes to the network and integrators of modules. These findings leverage new insights into how neuronal effective network topology relates to neuronal assembly self-organization mechanisms.

3D Bioprinted Neural-Like Tissue as a Platform to Study Neurotropism of Mouse-Adapted SARS-CoV-2.

The effects of neuroinvasion by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) become clinically relevant due to the numerous neurological symptoms observed in Corona Virus Disease 2019 (COVID-19) patients during infection and post-COVID syndrome or long COVID. This study reports the biofabrication of a 3D bioprinted neural-like tissue as a proof-of-concept platform for a more representative study of SARS-CoV-2 brain infection. Bioink is optimized regarding its biophysical properties and is mixed with murine neural cells to construct a 3D model of COVID-19 infection. Aiming to increase the specificity to murine cells, SARS-CoV-2 is mouse-adapted (MA-SARS-CoV-2) in vitro, in a protocol first reported here. MA-SARS-CoV-2 reveals mutations located at the Orf1a and Orf3a domains and is evolutionarily closer to the original Wuhan SARS-CoV-2 strain than SARS-CoV-2 used for adaptation. Remarkably, MA-SARS-CoV-2 shows high specificity to murine cells, which present distinct responses when cultured in 2D and 3D systems, regarding cell morphology, neuroinflammation, and virus titration. MA-SARS-CoV-2 represents a valuable tool in studies using animal models, and the 3D neural-like tissue serves as a powerful in vitro platform for modeling brain infection, contributing to the development of antivirals and new treatments for COVID-19.

3D Bioprinting of Murine Cortical Astrocytes for Engineering Neural-Like Tissue.

Astrocytes are glial cells with an essential role in the central nervous system (CNS), including neuronal support and functionality. These cells also respond to neural injuries and act to protect the tissue from degenerative events. In vitro studies of astrocytes' functionality are important to elucidate the mechanisms involved in such events and contribute to developing therapies to treat neurological disorders. This protocol describes a method to biofabricate a neural-like tissue structure rich in astrocytes by 3D bioprinting astrocytes-laden bioink. An extrusion-based 3D bioprinter was used in this work, and astrocytes were extracted from C57Bl/6 mice pups' brain cortices. The bioink was prepared by mixing cortical astrocytes from up to passage 3 to a biomaterial solution composed of gelatin, gelatin-methacryloyl (GelMA), and fibrinogen, supplemented with laminin, which presented optimal bioprinting conditions. The 3D bioprinting conditions minimized cell stress, contributing to the high viability of the astrocytes during the process, in which 74.08% ± 1.33% of cells were viable right after bioprinting. After 1 week of incubation, the viability of astrocytes significantly increased to 83.54% ± 3.00%, indicating that the 3D construct represents a suitable microenvironment for cell growth. The biomaterial composition allowed cell attachment and stimulated astrocytic behavior, with cells expressing the specific astrocytes marker glial fibrillary acidic protein (GFAP) and possessing typical astrocytic morphology. This reproducible protocol provides a valuable method to biofabricate 3D neural-like tissue rich in astrocytes that resembles cells' native microenvironment, useful to researchers that aim to understand astrocytes' functionality and their relation to the mechanisms involved in neurological diseases.

Notch1 and Galectin-3 Modulate Cortical Reactive Astrocyte Response After Brain Injury.

After a brain lesion, highly specialized cortical astrocytes react, supporting the closure or replacement of the damaged tissue, but fail to regulate neural plasticity. Growing evidence indicates that repair response leads astrocytes to reprogram, acquiring a partially restricted regenerative phenotype in vivo and neural stem cells (NSC) hallmarks in vitro. However, the molecular factors involved in astrocyte reactivity, the reparative response, and their relation to adult neurogenesis are poorly understood and remain an area of intense investigation in regenerative medicine. In this context, we addressed the role of Notch1 signaling and the effect of Galectin-3 (Gal3) as underlying molecular candidates involved in cortical astrocyte response to injury. Notch signaling is part of a specific neurogenic microenvironment that maintains NSC and neural progenitors, and Gal3 has a preferential spatial distribution across the cortex and has a central role in the proliferative capacity of reactive astrocytes. We report that in vitro scratch-reactivated cortical astrocytes from C57Bl/6J neonatal mice present nuclear Notch1 intracellular domain (NICD1), indicating Notch1 activation. Colocalization analysis revealed a subpopulation of reactive astrocytes at the lesion border with colocalized NICD1/Jagged1 complexes compared with astrocytes located far from the border. Moreover, we found that Gal3 increased intracellularly, in contrast to its extracellular localization in non-reactive astrocytes, and NICD1/Gal3 pattern distribution shifted from diffuse to vesicular upon astrocyte reactivation. In vitro, Gal3-/- reactive astrocytes showed abolished Notch1 signaling at the lesion core. Notch1 receptor, its ligands (Jagged1 and Delta-like1), and Hes5 target gene were upregulated in C57Bl/6J reactive astrocytes, but not in Gal3-/- reactive astrocytes. Finally, we report that Gal3-/- mice submitted to a traumatic brain injury model in the somatosensory cortex presented a disrupted response characterized by the reduced number of GFAP reactive astrocytes, with smaller cell body perimeter and decreased NICD1 presence at the lesion core. These results suggest that Gal3 might be essential to the proper activation of Notch signaling, facilitating the cleavage of Notch1 and nuclear translocation of NICD1 into the nucleus of reactive cortical astrocytes. Additionally, we hypothesize that reactive astrocyte response could be dependent on Notch1/Jagged1-Hes5 signaling activation following brain injury.

Neurorepair and Regeneration of the Brain: A Decade of Bioscaffolds and Engineered Microtissue.

Repairing the human brain remains a challenge, despite the advances in the knowledge of inflammatory response to injuries and the discovery of adult neurogenesis. After brain injury, the hostile microenvironment and the lack of structural support for neural cell repopulation, anchoring, and synapse formation reduce successful repair chances. In the past decade, we witnessed the rise of studies regarding bioscaffolds' use as support for neuro repair. A variety of natural and synthetic materials is available and have been used to replace damaged tissue. Bioscaffolds can assume different shapes and may or may not carry a diversity of content, such as stem cells, growth factors, exosomes, and si/miRNA that promote specific therapeutic effects and stimulate brain repair. The use of these external bioscaffolds and the creation of cell platforms provide the basis for tissue engineering. More recently, researchers were able to engineer brain organoids, neural networks, and even 3D printed neural tissue. The challenge in neural tissue engineering remains in the fabrication of scaffolds with precisely controlled topography and biochemical cues capable of directing and controlling neuronal cell fate. The purpose of this review is to highlight the existing research in the growing field of bioscaffolds' development and neural tissue engineering. Moreover, this review also draws attention to emerging possibilities and prospects in this field.

3D culture models to study SARS-CoV-2 infectivity and antiviral candidates: From spheroids to bioprinting.

The pandemic caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is receiving worldwide attention, due to the severity of the disease (COVID-19) that resulted in more than a million global deaths so far. The urgent need for vaccines and antiviral drugs is mobilizing the scientific community to develop strategies for studying the mechanisms of SARS-CoV-2 infection, replication kinetics, pathogenesis, host-virus interaction, and infection inhibition. In this work, we review the strategies of tissue engineering in the fabrication of three-dimensional (3D) models used in virology studies, which presented many advantages over conventional cell cultures, such as complex cytoarchitecture and a more physiological microenvironment. Scaffold-free (spheroids and organoids) and scaffold-based (3D scaffolding and 3D bioprinting) approach allow the biofabrication of more realistic models relevant to the pandemic, to be used as in vitro platforms for the development of new vaccines and therapies against COVID-19.

Neuroprotective effect of heparin Trisulfated disaccharide on ischemic stroke.

Cells undergoing hypoxia experience intense cytoplasmic calcium (Ca2+) overload. High concentrations of intracellular calcium ([Ca2+]i) can trigger cell death in the neural tissue, a hallmark of stroke. Neural Ca2+ homeostasis involves regulation by the Na+/Ca2+ exchanger (NCX). Previous data published by our group showed that a product of the enzymatic depolymerization of heparin by heparinase, the unsaturated trisulfated disaccharide (TD; ΔU, 2S-GlcNS, 6S), can accelerate Na+/Ca2+ exchange via NCX, in hepatocytes and aorta vascular smooth muscle cells. Thus, the objective of this work was to verify whether TD could act as a neuroprotective agent able to prevent neuronal cell death by reducing [Ca2+]i. Pretreatment of N2a cells with TD reduced [Ca2+]i rise induced by thapsigargin and increased cell viability under [Ca2+]I overload conditions and in hypoxia. Using a murine model of stroke, we observed that pretreatment with TD decreased cerebral infarct volume and cell death. However, when mice received KB-R7943, an NCX blocker, the neuroprotective effect of TD was abolished, strongly suggesting that this neuroprotection requires a functional NCX to happen. Thus, we propose TD-NCX as a new therapeutic axis for the prevention of neuronal death induced by [Ca2+]i overload.

Leptin enhances adult neurogenesis and reduces pathological features in a transgenic mouse model of Alzheimer's disease.

Alzheimer's disease (AD) is the most common dementia worldwide and is characterized by the presence of senile plaques by amyloid-beta (Aß) and neurofibrillary tangles of hyperphosphorylated Tau protein. These changes lead to progressive neuronal degeneration and dysfunction, resulting in severe brain atrophy and cognitive deficits. With the discovery that neurogenesis persists in the adult mammalian brain, including brain regions affected by AD, studies of the use of neural stem cells (NSCs) for the treatment of neurodegenerative diseases to repair or prevent neuronal cell loss have increased. Here we demonstrate that leptin administration increases the neurogenic process in the dentate gyrus of the hippocampus as well as in the subventricular zone of lateral ventricles of adult and aged mice. Chronic treatment with leptin increased NSCs proliferation with significant effects on proliferation and differentiation of newborn cells. The expression of the long form of the leptin receptor, LepRb, was detected in the neurogenic niches by reverse qPCR and immunohistochemistry. Moreover, leptin modulated astrogliosis, microglial cell number and the formation of senile plaques. Additionally, leptin led to attenuation of Aß-induced neurodegeneration and superoxide anion production as revealed by Fluoro-Jade B and dihydroethidium staining. Our study contributes to the understanding of the effects of leptin in the brain that may lead to the development of new therapies to treat Alzheimer's disease.

A novel decellularization method to produce brain scaffolds.

Scaffolds composed of extracellular matrix (ECM) can assist tissue remodeling and repair following injury. The ECM is a complex biomaterial composed of proteins, glycoproteins, proteoglycans, and glycosaminoglycans, secreted by cells. The ECM contains fundamental biological cues that modulate cell behavior and serves as a structural scaffold for cell adhesion and growth. For clinical applications, where immune rejection is a constraint, ECM can be processed using decellularization methods intended to remove cells and donor antigens from tissue or organs, while preserving native biological cues essential for cell growth and differentiation. Recent studies show bioengineered organs composed by a combination of a diversity of materials and stem cells as a possibility of new therapeutic strategies to treat diseases that affect different tissues and organs, including the central nervous system (CNS). Nevertheless, the methodologies currently described for brain decellularization involve the use of several chemical reagents with many steps that ultimately limit the process of organ or tissue recellularization. Here, we describe for the first time a fast and straightforward method for complete decellularization of mice brain by the combination of rapid freezing and thawing following the use of only one detergent (Sodium dodecyl sulfate (SDS)). Our data show that using the protocol we describe here, the brain was entirely decellularized, while still maintaining ECM components that are essential for cell survival on the scaffold. Our results also show the cell-loading of the decellularized brain matrix with Neuro2a cells, which were identified by immunohistochemistry in their undifferentiated form. We conclude that this novel and simple method for brain decellularization can be used as a scaffold for cell-loading.

Strategies to use fibrinogen as bioink for 3D bioprinting fibrin-based soft and hard tissues.

Fibrin gel has been widely used for engineering various types of tissues due to its biocompatible nature, biodegradability, and tunable mechanical and nanofibrous structural properties. Despite their promising regenerative capacity and extensive biocompatibility with various tissue types, fibrin-based biomaterials are often notoriously known as burdensome candidates for 3D biofabrication and bioprinting. The high viscosity of fibrin (crosslinked form) hinders proper ink extrusion, and its pre-polymer form, fibrinogen, is not capable of maintaining shape fidelity. To overcome these limitations and empower fibrinogen-based bioinks for fibrin biomimetics and regenerative applications, different strategies can be practiced. The aim of this review is to report the strategies that bring fabrication compatibility to these bioinks through mixing fibrinogen with printable biomaterials, using supporting bath supplemented with crosslinking agents, and crosslinking fibrin in situ. Moreover, the review discusses some of the recent advances in 3D bioprinting of biomimetic soft and hard tissues using fibrinogen-based bioinks, and highlights the impacts of these strategies on fibrin properties, its bioactivity, and the functionality of the consequent biomimetic tissue. Statement of Significance Due to its biocompatible nature, biodegradability, and tunable mechanical and nanofibrous structural properties, fibrin gel has been widely employed in tissue engineering and more recently, used as in 3D bioprinting. The fibrinogen's poor printable properties make it difficult to maintain the 3D shape of bioprinted constructs. Our work describes the strategies employed in tissue engineering to allow the 3D bioprinting of fibrinogen-based bioinks, such as the combination of fibrinogen with printable biomaterials, the in situ fibrin crosslinking, and the use of supporting bath supplemented with crosslinking agents. Further, this review discuss the application of 3D bioprinting technology to biofabricate fibrin-based soft and hard tissues for biomedical applications, and discuss current limitations and future of such in vitro models.

The Rise of Molecules Able To Regenerate the Central Nervous System.

Injury to the adult central nervous system (CNS) usually leads to permanent deficits of cognitive, sensory, and/or motor functions. The failure of axonal regeneration in the damaged CNS limits functional recovery. The lack of information concerning the biological mechanism of axonal regeneration and its complexity has delayed the process of drug discovery for many years compared to other drug classes. Starting in the early 2000s, the ability of many molecules to stimulate axonal regrowth was evaluated through automated screening techniques; many hits and some new mechanisms involved in axonal regeneration were identified. In this Perspective, we discuss the rise of the CNS regenerative drugs, the main biological techniques used to test these drug candidates, some of the most important screens performed so far, and the main challenges following the identification of a drug that is able to induce axonal regeneration in vivo.

Neuroprotective and restorative properties of the GLP-1/GIP dual agonist DA-JC1 compared with a GLP-1 single agonist in Alzheimer's disease.

The sister incretins glucagon-like peptide-1 (GLP-1) and glucagon dependent insulinotropic polypeptide (GIP) are growth factors responsible for re-sensitizing insulin signalling. Interestingly, their analogues, originally developed to treat type 2 diabetes (T2D), have demonstrated a range of neuroprotective and neurorestorative properties. Novel peptide GLP-1/GIP dual agonist (DA) shows good effects in diabetic patients, superior to the effects demonstrated by single GIP or GLP-1 mimetics. Furthermore, novel DAs have shown considerable neuroprotection in neurodegenerative models. Here, we investigated the neuroprotective and restorative involvement of the DA DA-JC1 and liraglutide (Lg), a single GLP-1 receptor analogue, in vitro using human neuroblastoma (SH-SY5Y) against oxidative stress induced by oxygen peroxide (H2O2), and in vivo, in a mouse model of Alzheimer's disease (AD), APP/PS1. First, we determined the ideal concentration of the peptides and demonstrated that DA-JC1 protects cells against oxidative stress more than Lg, improving cell viability, normalizing reactive oxygen species (ROS) and attenuating DNA damage generated by H2O2. Moreover, in 10-to-12-months-old APP/PS1 animals treated for 4 weeks, both Lg and DA-JC1 were very efficient in stimulating neurogenesis and reducing some important hallmarks of AD, but DA-JC1 was better than Lg in attenuating crucial neuroinflammatory markers, especially reactive astrocyte, in both wild-type (WT) and APP/PS1 hippocampal regions. Altogether, this study suggests an interactive role of GLP-1 and GIP receptors, enhancing the efficiency of single GLP-1 analogues, especially in attenuating oxidative stress and neuroinflammation. We confirm that combining GLP-1 and GIP results in a variety of beneficial effects, providing key evidences for the development of a promising therapeutic strategy for AD.