The mood-improving actions of antidepressants do not depend on neurogenesis but are associated with neuronal remodeling

The mechanisms underlying the initiation/onset of, and the recovery from, depression are still largely unknown; views that neurogenesis in the hippocampus may be important for the pathogenesis and amelioration of depressive symptoms have gained currency over the years although the original evidence has been challenged. In this study, an unpredictable chronic mild stress protocol was used to induce a depressive-like phenotype in rats. In the last 2 weeks of stress exposure, animals were treated with the antidepressants fluoxetine, imipramine, CP 156,526 or SSR 1494515, alone or combined with methylazoxymethanol, a cytostatic agent used to arrest neurogenesis. We found that antidepressants retain their therapeutic efficacy in reducing both measured indices of depression-like behavior (learned helplessness and anhedonia), even when neurogenesis is blocked. Instead, our experiments suggest re-establishment of neuronal plasticity (dendritic remodeling and synaptic contacts) in the hippocampus and prefrontal cortex, rather than neurogenesis, as the basis for the restoration of behavioral homeostasis by antidepressants.This project used compound(s) provided by the National Cancer Institute’s Chemical Carcinogen Reference Standards Repository (operated under contract N02-CB-07008 by Midwest Research Institute; MAM), Sanofi-Synthelabo (SSR 149415) and Pfizer (CP 156,526). The authors’ work was supported by the Portuguese Foundation for Science and Technology (FCT) (PTDC/SAU-NEU/72699/2006)

Microparticles loaded gellan gum hydrogel matrices: engineering tissues for nucleus pulposus regeneration

The intervertebral disc central core is made by a gel-like tissue structure composed of more than 80% of water, Nucleus Pulposus (NP). Proteoglycans such as versican and especially aggrecan are the main constituents of the NP matrix as well as collagen type II. The purpose of this work is creating novel Gellan gum-based (GG) hydrogel formulations. GG microparticles (MPs) dispersed in a GG matrix are the novelty for finding application as NP substitute. The ongoing experiment comprises de GG functionalization through methacrylated groups addition. In order to optimize some properties of GG, the functionalization will allow us to improve the water solubility and photopolymerization in situ of the biomaterial. High acyl (HA) and Low acyl (LA) Gellan Gum (GG) at different ratio [75%:25% (v/v); 50%:50% (v/v), 25%:75% (v/v)], HAGG 0.75% and LAGG 2%, were mixed in order to prepare solutions to be used as formulations of GG MPs/hydrogels matrix. The GG MP/hydrogel matrix formulations were characterized by dynamic mechanical analysis (DMA), swelling behaviour and degradation rate. The toxic effect of GG MPs/hydrogel discs leachables onto the cells was investigated in vitro using a mouse lung fibroblast-like cell (L929 cells) line. Live/Dead cell viability assay was performed to assess the encapsulation efficacy; meanwhile DAPI/Phalloidin staining was performed to evaluate cell morphology. The Methacrylated Gellan Gum (GG-MA) was prepared following the protocol [1]. Mechanism reactions occurred in presence of glycidyl methacrylate by addition to a solution of LAGG at 2% (w/v). The reaction was running over 24 h at room temperature controlling the pH at 8.5 with sodium hydroxide 1 M. GG MPs size was measured using a stereo microscope by staining the MPs with Toluidine Blue-O. This method also allowed evaluating the MPs dispersion and matrix cohesion. From DMA analysis it was observed that the range of 50–500 mg/mL of incorporated MPs is the optimal concentration to reinforce GG matrices. It was demonstrated the non–cytotoxic effect of MPs/hydrogels over L929 cells. In fact, L929 cells were successfully encapsulated in all GG formulations GG MPs and remaining viable over 72 h of culturing. The resulting product from methacrylation reaction was evaluated by nuclear magnetic resonance to assess the reaction efficiency and the degree of substitution. Methacrylated Gellan gum and GG MP/hydrogel matrix are promising hydrogels to be used in tissue engineering strategies for treatment of the degenerated NP

Injectable gellan gum-based hydrogels for intervertebral disc regeneration

Intervertebral disc (IVD) degeneration is a challenging pathology that, due to the inefficiency of the current treatments, urgently demands for the development of new regenerative approaches[1]. The best viable implant material for nucleus pulposus (NP) regeneration has yet to be identified, but it is believed that biodegradable hydrogelbased materials are promising candidates[2]. In this work, we are proposing the use of ionic- and photocrosslinked methacrylated gellan gum (GG-MA) hydrogels as potential acellular and cellular injectable scaffolds for IVD regeneration

Gellan gum-based hydrogels for intervertebral disc tissue engineering applications

Intervertebral disc (IVD) degeneration is a challenging clinical problem that urgently demands viable nucleus pulposus (NP) implant materials. The best suited biomaterial for NP regeneration has yet to be identified, but it is believed that biodegradable hydrogel-based materials are promising candidates. In this work, we have developed ionic- and photo-crosslinked methacrylated gellan gum (GG–MA) hydrogels to be used in acellular and cellular tissue-engineering strategies for the regeneration of IVDs. The physicochemical properties of the developed hydrogels were investigated by Fourier-transform infrared spectroscopy, 1H nuclear magnetic resonance and differential scanning calorimetry. The swelling ability and degradation rate of hydrogels were also analysed in phosphate-buffered saline solution at physiological pH for a period of 30 days. Additionally, the morphology and mechanical properties of the hydrogels were assessed under a scanning electron microscope and dynamic compression, respectively. An in vitro study was carried out to screen possible cytotoxicity of the gellan gum-based hydrogels by culturing rat lung fibroblasts (L929 cells) with hydrogel leachables up to 7 days. The results demonstrated that gellan gum was successfully methacrylated. We observed that the produced GG–MA hydrogels possess improved mechanical properties and lower water uptake ability and degradation rate as compared to gellan gum. This work also revealed that GG–MA hydrogels are non-cytotoxic in vitro, thus being promising biomaterials to be used in IVD tissue-engineering strategies.The authors are grateful for funds provided by the Portuguese Foundation for Science and Technology (FCT) through the POCTI and FEDER programmes, including Project ProteoLight (Grant No. PTDC/FIS/68517/2006). This work was also carried outwith the support of the European Union-funded Collaborative Project Disc Regeneration (Grant No. NMP3-LA-2008-213904)

Development of gellan gum-based microparticles/hydrogel matrices for application in the intervertebral disc regeneration

Low back pain is one of the most reported medical conditions associated to intervertebral disc (IVD) degeneration. Nucleus pulposus (NP) is often regarded as the structure where intervertebral disc degeneration begins. Gellan gum-based (GG) hydrogels for acellular and cellular tissue engineering strategies have been developed for finding applications as NP substitutes. The innovative strategy is based on the reinforcement of the hydrogel matrix with biocompatible and biodegradable GG microparticles (MPs), which are expected to improve the mechanical properties, while allowing to tailor its degradation rate. In this study, several GG MPs/hydrogels discs formulations were prepared by means of mixing high (HAGG 0.75% (w/v)) and low acyl (LAGG 2% (w/v)) GG aqueous solutions at different ratios, namely 75%:25% (v/v), 50%:50% (v/v), 25%:75% (v/v), respectively. The GG MPs size was measured using a stereo microscope and their dispersion within the hydrogel matrix was evaluated by means of staining the MPs with Toluidine Blue-O. The developed GG MPs/hydrogel discs were physico-chemically characterized by Fourier-transform infrared spectroscopy and 1H-nuclear magnetic resonance spectroscopy. The swelling behaviour and degradation rate were assessed by immersion in a phosphate buffer saline solution for the period of 14 days. The morphology and mechanical behaviour were investigated by scanning electron microscopy and dynamic mechanical analysis, respectively. The mechanical properties of the hydrogels discs were improved by mixing the gels with the MPs. In addition, the possible cytotoxicity of the leachables released by MPs/hydrogel discs was screened in vitro, using a mouse lung fibroblast cell line (L929 cells). In order to investigate the encapsulation efficacy of L929 cells into the GG MPs/hydrogel discs, cells were stained with DAPI blue/Texas Red-Phalloidin and observed by confocal microscopy, after 24, 48 and 72 hours of culturing. A cell viability assay was also performed using Calcein AM staining. The cell culture studies demonstrated that MPs/hydrogel discs are non-cytotoxic over L929 cells. It was also demonstrated that L929 cells can be successfully encapsulated into the GG MPs of different formulations, remaining viable after 72 hours of culturing. This study showed that GG hydrogel matrices reinforced with cell-loaded MPs could be a candidate strategy for NP regeneration.Fundação para a Ciência e a Tecnologia (FCT) throught the POCTI and FEDER, including ProteoLigh

A Trans-Dimensional Approach to the Behavioral Aspects of Depression

Depression, a complex mood disorder, displays high comorbidity with anxiety and cognitive disorders. To establish the extent of inter-dependence between these behavioral domains, we here undertook a systematic analysis to establish interactions between mood [assessed with the forced-swimming (FST) and sucrose consumption tests (SCT)], anxiety [elevated-plus maze (EPM) and novelty suppressed feeding (NSF) tests] and cognition (spatial memory and behavioral flexibility tests) in rats exposed to unpredictable chronic-mild-stress (uCMS). Expectedly, uCMS induced depressive-like behavior, a hyperanxious phenotype and cognitive impairment; with the exception of the measure of anxiety in the EPM, these effects were attenuated by antidepressants (imipramine, fluoxetine). Measures of mood by the FST and SCT were strongly correlated, whereas no significant correlations were found between the different measures of anxiety (EPM and NSF); likewise, measures of cognition by spatial memory and behavioral flexibility tests were poorly correlated. Inter-domain analysis revealed significant correlations between mood (FST and SCT) and anxiety-like behavior (NSF, but not EPM). Furthermore, significant correlations were found between cognitive performance (reverse learning task) and mood (FST and SCT) and anxiety-like behavior (NSF). These results demonstrate interactions between different behavioral domains that crosscut the disciplines of psychiatry and neurology

Peripheral axonal ensheathment is regulated by RalA GTPase and the exocyst complex

Funding This work was supported by H2020 Marie Skłodowska-Curie Actions [H2020- GA661543-Neuronal Trafficking to R.O.T.], Fundo Regional para a Ciência e Tecnologia [IF/00392/2013/CP1192/CT0002 to R.O.T.] and iNOVA4Health (UID/Multi/04462/2013) (co-funded by FCT-FEDER-PT2020).Axon ensheathment is fundamental for fast impulse conduction and the normal physiological functioning of the nervous system. Defects in axonal insulation lead to debilitating conditions, but, despite its importance, the molecular players responsible are poorly defined. Here, we identify RalA GTPase as a key player in axon ensheathment in Drosophila larval peripheral nerves. We demonstrate through genetic analysis that RalA action through the exocyst complex is required in wrapping glial cells to regulate their growth and development. We suggest that the RalA-exocyst pathway controls the targeting of secretory vesicles for membrane growth or for the secretion of a wrapping glia-derived factor that itself regulates growth. In summary, our findings provide a new molecular understanding of the process by which axons are ensheathed in vivo, a process that is crucial for normal neuronal function.publishersversionpublishe

Bioactive macro/micro porous silk fibroin/Nano-sized calcium phosphate scaffolds with potential for bone tissue engineering applications

Aim: The development of novel silk/nano-sized calcium phosphate (silk/nano-CaP) scaffolds with highly dispersed CaP nanoparticles in the silk fibroin (SF) matrix for bone tissue engineering. Materials & methods: Nano-CaP was incorporated in a concentrated aqueous SF solution (16 wt.%) by using an in situ synthesis method. The silk/nano-CaP scaffolds were then prepared through a combination of salt-leaching/ lyophilization approaches. Results: The CaP particles presented good affinity to SF and their size was inferior to 200 nm when theoretical CaP/silk ratios were between 4 and 16 wt.%, as determined by scanning electron microscopy. The CaP particles displayed a uniform distribution in the scaffolds at both microscopic and macroscopic scales as observed by backscattered scanning electron microscopy and micro-computed tomography, respectively. The prepared scaffolds presented self-mineralization capability and no cytotoxicity confirmed by in vitro bioactivity tests and cell viability assays, respectively. Conclusion: These results indicated that the produced silk/nano-CaP scaffolds could be suitable candidates for bone-tissueengineering applications.This study was funded by the Portuguese Foundation for Science and Technology (FCT) through the projects Tissue2Tissue (PTDC/CTM/105703/2008) and Osteo Cart (PTDC/CTM-BPC/115977/2009). The funding from Foundation Luso-Americana is greatly acknowledged. L-P Yan gives thanks for his PhD scholarship from FCT (SFRHIBD/64717/2009). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or potions, expert testimony, grants or patents received or pending, or royalties