Microneurotrophin BNN27 Reduces Astrogliosis and Increases Density of Neurons and Implanted Neural Stem Cell-Derived Cells after Spinal Cord Injury.

Microneurotrophins, small-molecule mimetics of endogenous neurotrophins, have demonstrated significant therapeutic effects on various animal models of neurological diseases. Nevertheless, their effects on central nervous system injuries remain unknown. Herein, we evaluate the effects of microneurotrophin BNN27, an NGF analog, in the mouse dorsal column crush spinal cord injury (SCI) model. BNN27 was delivered systemically either by itself or combined with neural stem cell (NSC)-seeded collagen-based scaffold grafts, demonstrated recently to improve locomotion performance in the same SCI model. Data validate the ability of NSC-seeded grafts to enhance locomotion recovery, neuronal cell integration with surrounding tissues, axonal elongation and angiogenesis. Our findings also show that systemic administration of BNN27 significantly reduced astrogliosis and increased neuron density in mice SCI lesion sites at 12 weeks post injury. Furthermore, when BNN27 administration was combined with NSC-seeded PCS grafts, BNN27 increased the density of survived implanted NSC-derived cells, possibly addressing a major challenge of NSC-based SCI treatments. In conclusion, this study provides evidence that small-molecule mimetics of endogenous neurotrophins can contribute to effective combinatorial treatments for SCI, by simultaneously regulating key events of SCI and supporting grafted cell therapies in the lesion site.

Development and Comparative In Vitro and In Vivo Study of BNN27 Mucoadhesive Liposomes and Nanoemulsions for Nose-to-Brain Delivery.

Intranasal administration offers an alternative and promising approach for direct nose-to-brain delivery. Herein, we developed two chitosan (CHT)-coated (and uncoated) nanoformulations of BNN27 (a synthetic C-17-spiro-dehydroepiandrosterone analogue), liposomes (LIPs), and nanoemulsions (NEs), and compared their properties and brain disposition (in vitro and in vivo). LIPs were formulated by thin film hydration and coated with CHT by dropwise addition. BNN27-loaded NEs (BNEs) were developed by spontaneous emulsification and optimized for stability and mucoadhesive properties. Mucoadhesive properties were evaluated by mucin adherence. Negatively charged CHT-coated LIPs (with 0.1% CHT/lipid) demonstrated the highest coating efficiency and mucoadhesion. BNEs containing 10% w/w Capmul-MCM and 0.3% w/w CHT demonstrated the optimal properties. Transport of LIP or NE-associated rhodamine-lipid across the blood-brain barrier (in vitro) was significantly higher for NEs compared to LIPs, and the CHT coating demonstrated a negative effect on transport. However, the CHT-coated BNEs demonstrated higher and faster in vivo brain disposition following intranasal administration compared to CHT-LIPs. For both BNEs and LIPs, CHT-coating resulted in the increased (in vivo) brain disposition of BNN27. Current results prove that CHT-coated NEs consisting of compatible nasal administration ingredients succeeded in to delivering more BNN27 to the brain (and faster) compared to the CHT-coated LIPs.

Interfacing reduced graphene oxide with an adipose-derived extracellular matrix as a regulating milieu for neural tissue engineering.

Enthralling evidence of the potential of graphene-based materials for neural tissue engineering is motivating the development of scaffolds using various structures related to graphene such as graphene oxide (GO) or its reduced form. Here, we investigated a strategy based on reduced graphene oxide (rGO) combined with a decellularized extracellular matrix from adipose tissue (adECM), which is still unexplored for neural repair and regeneration. Scaffolds containing up to 50 wt% rGO relative to adECM were prepared by thermally induced phase separation assisted by carbodiimide (EDC) crosslinking. Using partially reduced GO enables fine-tuning of the structural interaction between rGO and adECM. As the concentration of rGO increased, non-covalent bonding gradually prevailed over EDC-induced covalent conjugation with the adECM. Edge-to-edge aggregation of rGO favours adECM to act as a biomolecular physical crosslinker to rGO, leading to the softening of the scaffolds. The unique biochemistry of adECM allows neural stem cells to adhere and grow. Importantly, high rGO concentrations directly control cell fate by inducing the differentiation of both NE-4C cells and embryonic neural progenitor cells into neurons. Furthermore, primary astrocyte fate is also modulated as increasing rGO boosts the expression of reactivity markers while unaltering the expression of scar-forming ones.

Neural stem cell delivery via porous collagen scaffolds promotes neuronal differentiation and locomotion recovery in spinal cord injury.

Neural stem cell (NSC) grafts have demonstrated significant effects in animal models of spinal cord injury (SCI), yet their clinical translation remains challenging. Significant evidence suggests that the supporting matrix of NSC grafts has a crucial role in regulating NSC effects. Here we demonstrate that grafts based on porous collagen-based scaffolds (PCSs), similar to biomaterials utilized clinically in induced regeneration, can deliver and protect embryonic NSCs at SCI sites, leading to significant improvement in locomotion recovery in an experimental mouse SCI model, so that 12 weeks post-injury locomotion performance of implanted animals does not statistically differ from that of uninjured control animals. NSC-seeded PCS grafts can modulate key processes required to induce regeneration in SCI lesions including enhancing NSC neuronal differentiation and functional integration in vivo, enabling robust axonal elongation, and reducing astrogliosis. Our findings suggest that the efficacy and translational potential of emerging NSC-based SCI therapies could be enhanced by delivering NSC via scaffolds derived from well-characterized clinically proven PCS.

Neurosteroids as regulators of neuroinflammation.

Neuroinflammation is a physiological protective response in the context of infection and injury. However, neuroinflammation, especially if chronic, may also drive neurodegeneration. Neurodegenerative diseases, such as multiple sclerosis (MS), Alzheimer's disease (AD), Parkinson's disease (PD) and traumatic brain injury (TBI), display inflammatory activation of microglia and astrocytes. Intriguingly, the central nervous system (CNS) is a highly steroidogenic environment synthesizing steroids de novo, as well as metabolizing steroids deriving from the circulation. Neurosteroid synthesis can be substantially affected by neuroinflammation, while, in turn, several steroids, such as 17ß-estradiol, dehydroepiandrosterone (DHEA) and allopregnanolone, can regulate neuroinflammatory responses. Here, we review the role of neurosteroids in neuroinflammation in the context of MS, AD, PD and TBI and describe underlying molecular mechanisms. Moreover, we introduce the concept that synthetic neurosteroid analogues could be potentially utilized for the treatment of neurodegenerative diseases in the future.

Controlling the Outgrowth and Functions of Neural Stem Cells: The Effect of Surface Topography.

Neural stem cells (NSCs) are self-renewing cells that generate the major cell types of the central nervous system, namely neurons, astrocytes and oligodendrocytes, during embryonic development and in the adult brain. NSCs reside in a complex niche where they are exposed to a plethora of signals, including both soluble and physical signals such as compressive and shear stresses, but also discontinuities and differences in morphology of the extracellular environment, termed as topographical features. Different approaches that incorporate artificial micro- and nano-scale surface topographical features have been developed aiming to recapitulate the in vivo NSC niche discontinuities and features, particularly for in vitro studies. The present review article aims at reviewing the existing body of literature on the use of artificial micro- and nano-topographical features to control NSCs orientation and differentiation into neuronal and/or neuroglial lineage. The different approaches on the study of the underlying mechanism of the topography-guided NSC responses are additionally revised and discussed.

Expression of the epidermal growth factor receptor (EGFR) and the phosphorylated EGFR in invasive breast carcinomas.

INTRODUCTION: Epidermal growth factor receptor (EGFR) is involved in regulating cell growth in breast carcinomas. Its activated form, phosphorylated EGFR (pEGFR), is correlated with poor prognosis in lung cancer, but it has not yet been fully investigated in breast cancer. The aim of this study was to investigate the expressions of EGFR and pEGFR and their correlation with overall and disease-free survival, clinicopathological parameters and biological markers of invasion and angiogenesis (phosphorylated Akt [pAkt], urokinase plasminogen activator receptor [uPAR], matrix metalloproteinase [MMP]-14, vascular endothelial growth factor receptor [VEGFR]-1/Flt-1). METHODS: A three-step immunohistochemical method was applied to paraffin-embedded sections from 154 patients with invasive breast carcinoma in order to detect expressions of the proteins EGFR, pEGFR, oestrogen receptor, progesterone receptor, c-erbB-2, pAkt, VEGFR-1/Flt-1, MMP-14 and uPAR. The results were evaluated statistically using the chi2 test. Overall and disease-free survival distribution curves were assessed using the Kaplan-Meier test and log-rank statistics, followed by Cox proportional hazards regression model. RESULTS: EGFR and pEGFR proteins were immunodetected in the membrane of the malignant cells (11.3% and 35.7%, respectively). EGFR expression was positively correlated with nuclear grade (P = 0.001) and negatively correlated with the hormonal receptor oestrogen receptor (P = 0.005). pEGFR was positively related to the Akt pathway (P = 0.008) and appeared to participate in invasion and metastasis (uPAR, P = 0.049; MMP-14, P = 0.025; VEGFR-1/Flt-1, P = 0.016). Univariate analysis showed that the EGFR/pEGFR phenotype was associated with poor overall survival (P = 0.019), a finding further supported by multivariate analysis (P = 0.013). CONCLUSION: These data provide evidence that pEGFR expression is related to angiogenesis (via VEGFR-1/Flt-1, MMP-14 and pAkt pathways) and invasiveness (via uPAR, MMP-14 and pAkt pathways) and that the EGFR/pEGFR phenotype is associated with poor patient survival in invasive breast cancer.