Academic literature on the topic 'Organotypic spinal cultures'

Objective:Reports about the role of autoimmunity in amyotrophic lateral sclerosis (ALS) are inconsistent. The aim of this work was to investigate the effect of IgG from patients with ALS on motor neurons in a physiological-like surrounding.Methods:Using affinity chromatography, IgG from six ALS patients, four disease controls and five healthy subjects was purified. Organotypic spinal cord cultures, which conserve the structure of the spinal cord in a horizontal plane and are suitable for studies with long-term treatment, were used and IgG with different concentrations ranging from 0.05 mg/mL to 0.5 mg/mL was added to the culture medium. Ventral motor neuron survival was evaluated by morphology and SMI-32 immunohistochemistry staining. Lactate dehydrogenase (LDH) level in the culture medium was measured by colorimetry.Results:After cultures were treated with ALS IgG for three weeks, the number and morphology of motor neurons showed little change. In addition, there was no significant difference in lactate dehydrogenase release between cultures treated with medium alone, normal control IgG, disease control IgG or ALS IgG.Conclusions:The results indicate that IgG from these ALS patients was insufficient per se to induce motor neuron death in Organotypic slice cultures. However, this does not preclude the possibility that other changes may have occurred in the motor neurons. This work offered a new model to evaluate the role of IgG in the pathogenesis of ALS. Organotypic cultures contribute to study of the impact of IgG on motor neurons by mimicking physiological conditions.

Recent studies suggest ex vivo modeling of neuronal injury is a robust approach for the mechanistic study of neurodegeneration. Melatonin, an indolamine, is a versatile molecule with antioxidative, antiapoptotic, neuroprotective, and anti-inflammatory properties. While melatonin has been studied as a therapeutic agent for spinal cord injury (SCI) related neuronal cell loss, its actions in organotypic slice cultures approximating SCI effects are less well understood. The actions of melatonin were therefore examined following exposure of cultured rat spinal cord slices to glutamate excitotoxicity. Exposure to glutamate (500 μM) for 4 hours induced neuronal degeneration that was prevented by 0.5 μM melatonin (applied immediately or 4 hours following glutamate exposure). Decreased internucleosomal DNA fragmentation, Bax:Bcl-2 and calpain:calpastatin ratios, caspase 8, 9 and 3 activities in slice cultures were measured following melatonin treatment. Melatonin receptor (MTR1, MTR2) mRNA levels were increased in the melatonin treated spinal cord slices. To confirm melatonin receptor-mediated protection, slice cultures were treated with 10 or 25 μM luzindole (melatonin receptor antagonist) at 0 and 4 hours, respectively, after glutamate exposure. Luzindole significantly decreased the ability of melatonin to prevent cell death in the sliced culture model. These results suggest melatonin receptors may provide a pathway for therapeutic applications to prevent penumbral neuron loss following SCI.

2012/2013
My PhD project concerns neurodegenerative processes in the mouse spinal cord, with a particular attention to amyotrophic lateral sclerosis (ALS). During my PhD I have used as a model the organotypic spinal slice cultures and I have focused my studies on: early changes in spinal tissue excitability in an ALS genetic model; spinal network activity changes induced by oxidative stress in wild type (WT) and synaptic activity in premotor circuits when challenged by neuroinflammation in WT. The principal aim of my work was to understand the dialogue between a general stress condition and the spinal premotor network. To this aim, I combined electrophysiological techniques and immunofluorescence analysis to characterized the ventral interneurones located in spinal microcircuits. For that purpose I exploited the organotypic cultures developed from embryonic mouse spinal cord that are generally accepted as a good model to study the neuronal premotor activity and provide high experimental access to interneurones (Avossa et al., 2003). In the first part of my research I compared WT cultures with SOD1G93A transgenic cultures, one of the more investigated ALS model. In cultured spinal networks, as described in acute preparation collected at different stages of development, there is a progressive fastening of glycinergic currents, represented by the reduction of the decay time constant (tau value) of synaptic currents along with the slices growth. WT and SOD1G93A cultures display a different maturation profile since in transgenic slices this developmental process is significantly steeper not only in glycinergic post synaptic currents (PSCs) but also in miniature PSCs (mPSCs). This difference in the glycinergic PSCs kinetic properties can be strongly reduced by the presence of TBOA that lowers the GABA synthesis. These results support the hypothesis that in SOD1G93A cultures there is an increase amount of glycine and GABA co-release leading to the conclusion that the synaptic release is conditioned by the presence of the mutation at an early stage of development, before any evident neuronal degeneration. Moreover, I supported this data also with preliminary results regarding the co-staining of GlyT2 and GAD65 (markers for presynaptic glycine and GABA, respectively). In fact, SOD1G93A spinal organotypic slices seem to display an higher amount of mixed synapses. Next, I tested other stress processes of the tissue that could potentially affect synaptic activity and, ultimately, alter network activity. I tested chronic incubations of the spinal slices, since they are long-term preparations, with stress key players: hydrogen peroxide (H2O2) to create an oxidative stress and lipopolysaccharide (LPS) or a mixture of cytokines (CKs: TNF-α, IL-1β and GM-CSF) to mimic neuroinflammation. For these sets of experiments I have used another strain of mice with no genetic manipulation. All these chronic treatments increase the AMPA receptor mediated PSCs frequency; moreover, a neuroinflammation state is able to enhance the overall network activity; LPS treatment increases also the amplitude of AMPA-mediated synaptic currents in both spontaneous and miniature events, while CKs accelerate the disinhibited burst rhythm induced by the pharmacological removal of the synaptic inhibition that switch on the rhythmogenic centre contained in the spinal network. Summarizing, I detected that the treatments with these environmental cues affected the synaptic component, in this case the excitatory one, of the premotor network. Altogether, my work highlighted that a genetic predisposition (in the case of familial ALS) and environmental factors of different kind (oxidative and inflammatory factors or an alterate SOD1) trigger changes in synaptic transmission and we may speculate that these alterations in the premotor circuit could cooperate in synergy leading to the development of misconnected networks that contribute to induce motoneuronal neurodegeneration. Riassunto Il mio progetto di dottorato riguarda processi neurodegenerativi del midollo spinale, con una particolare attenzione verso la sclerosi laterale amiotrofica (SLA). Durante il mio dottorato ho usato come modello le fettine organotipiche di midollo spinale e ho concentrato i miei studi su: cambiamenti precoci nell’eccitabilità del tessuto spinale in un modello genetico di SLA; cambiamenti nell’attività del network spinale indotti da stress ossidativo in wild type (WT) e cambiamenti nell’attività sinaptica dei circuiti premotori sottoposti ad uno stress infiammatorio in WT. Il fine principale del mio lavoro era quello di capire il dialogo tra una condizione di stress generale ad il network spinale premotorio. A questo scopo, ho unito tecniche elettrofisiologiche e analisi di immunofluorescenza per caratterizzare gli interneuroni ventrali localizzati nel microcircuito spinale. Per raggiungere questo obiettivo ho sfruttato le colture organotipiche derivate dal midollo spinale di embrioni di topo che sono generalmente accettate come un buon modello per studiare l’attività premotoria neuronale e garantiscono un facile accesso sperimentale agli interneuroni (Avossa et al., 2003). Nella prima parte della mia ricerca ho confrontato colture WT con colture transgeniche SOD1G93A, uno dei modelli di SLA maggiormente studiati. Nei network spinali in coltura, come già descritto in preparazioni acute ottenute a diversi stadi di sviluppo, c’è una progressiva velocizzazione delle correnti glicinergiche, rappresentata dalla riduzione del decay time constant (valore di tau) delle correnti sinaptiche durante la crescita delle fettine. Le colture WT e SOD1G93A presentano un diverso profilo di maturazione dato che nelle colture transgeniche questo processo di sviluppo è significativamente più marcato non solo nelle correnti postsinaptiche (PSCs) ma anche negli eventi in miniatura (mPSCs). Questa differenza nelle proprietà cinetiche delle correnti glicinergiche può essere fortemente ridotta dalla presenza di TBOA che diminuisce la sintesi del GABA. Questi risultati supportano l’ipotesi che nelle colture SOD1G93A ci sia un aumento del co-rilascio GABA/glicina portando alla conclusione che il rilascio sinaptico sia condizionato dalla presenza della mutazione ad uno stadio precoce dello sviluppo, prima di qualsiasi degenerazione neuronale evidente. Inoltre, ho supportato questo dato anche con risultati preliminari riguardanti la marcatura di GlyT2 e GAD65 (due marker per la glicina ed il GABA presinaptici rispettivamente). Infatti, le fettine spinali organotipiche SOD1G93A sembrano caratterizzate da un aumento delle sinapsi miste. Successivamente, ho testato altri processi di stress del tessuto che potrebbero interferire con l’attività sinaptica e, conseguentemente, alterare l’attività del network. Dato che le fettine spinali sono preparazioni a lungo termine, ho testato incubazioni croniche con molecole chiave nei processi di stress: perossido di idrogeno (H2O2) per creare uno stress ossidativo e lipopolisaccaride (LPS) o una miscela di citochine (CKs: TNF-α, IL-1β and GM-CSF) per mimare uno stato infiammatorio. Per questo set di esperimenti ho usato un altro ceppo di topi privo di manipolazione genetica. Tutti questi trattamenti cronici aumentano la frequenza delle correnti mediate dai recettori AMPA; inoltre, uno stato infiammatorio è in grado di incrementare l’attività globale del network; il trattamento con LPS aumenta anche l’ampiezza delle correnti sinaptiche AMPA-mediate, sia spontanee che in miniatura, mentre le CKs accelerano il ritmo dei burst indotto dall’eliminazione farmacologica dell’inibizione sinaptica che accende il centro ritmogenico presente nel network spinale. Riassumendo, ho dimostrato che i trattamenti con questi fattori ambientali alterano la componente sinaptica, in questo caso eccitatoria, del network premotorio. Nel complesso il mio lavoro ha evidenziato che una predisposizione genetica (nel caso della SLA familiare) e fattori ambientali di varia natura (ossidativi, infiammatori o di alterata SOD1) inducono cambiamenti nella trasmissione sinaptica e possiamo speculare sul fatto che queste alterazioni nel circuito premotorio possono cooperare in sinergia causando lo sviluppo di network inefficienti che concorrono a determinare la neurodegenerazione motoneuronale.
XXVI Ciclo
1985

2009/2010
Amyotrophic lateral sclerosis (ALS) is a lethal neurodegenerative disease characterized by loss of motoneurons. The discovery of mutations in the gene for the cytosolic Cu/Zn superoxide dismutase in a small proportion of familiar ALS patients led to an animal model in which the human mutant SOD1 is overexpressed in mice (G93A). For this study, we employed the long term spinal cord organotypic cultures developed from G93A embryonic mice and their wild type (WT) littermates, starting from the recent findings emerged from a study by Avossa et al. (2006). These authors reported that G93A organotypic spinal cultures exhibited increased vulnerability to AMPA glutamate receptormediated excitotoxic stress, prior to clear disease appearance, besides showing a significantly increased ratio between inhibitory and excitatory synapses, although they did not express evident morphological differences, when compared to WT ones (Avossa et al., 2006). The primary objective of this study was to investigate this early ALS stage to understand how functional changes can predate morphological alterations. To that aim we monitored spontaneous synaptic activity via patch clamping interneurons both in WT and G93A spinal cultures after 7, 14 and 21 days of in vitro (DIV) growth. At 7 DIV, when synchronous episodes of activity are normally detected in cultured spinal circuits, G93A slices displayed bursting with a higher probability (83%) when compared to controls (54%). Between 14 and 21 DIV, when bursting activity disappear, both in G93A and WT slices, pharmacological dissection of glutamate, GABA and glycine mediated post synaptic currents (PSCs), showed, in G93A, a significant reduction in GABAergic PSCs and mPSCs in respect to WT. Upon pharmacological removal of the GABAergic component, fast glycinergic events were unmasked and these events displayed a similar frequency in both culture groups. Along with in vitro growth, we detected a progressive reduction in the decay time constant of glycinergic PSCs, such process was significantly faster in G93A. Thus, a shift in dynamic communication within spinal networks might be involved in ALS progression.
XXIII Ciclo
1980

2007/2008
Nel midollo spinale lo sviluppo in circuiti funzionali delle reti neuronali dell’area ventrale è un processo complesso, che coinvolge meccanismi genetici ed epigenetici che promuovono la maturazione del controllo motorio (Jessell, 2000; Kiehn, 2006). Far luce su tali meccanismi è un passo cruciale per identificare quei neuroni che risultano essere più vulnerabili in caso di patologie degenerative del midollo spinale, ma anche per elaborare nuove strategie nel campo della rigenerazione dei circuiti danneggiati. Le colture organotipiche ottenute dal midollo spinale embrionale di topo e mantenute in vitro per 1 o 2 settimane, riepilogano molti dei processi che caratterizzano lo sviluppo dei segmenti spinali in vivo e sono particolarmente adatte allo studio della maturazione della rete spinale (Avossa et al., 2003; Rosato-Siri et al., 2004; Furlan et al., 2005; Furlan et al., 2007). In questa tesi ho usato tale modello per studiare, nei segmenti di midollo spinale embrionale, il controllo spazio-temporale di segnali intracellulari al Ca2+, generati da popolazioni neuronali appartenenti ai circuiti motori. Ho osservato la presenza di segnali ripetuti al Ca2+ dipendenti dall’età delle colture, monitorando le dinamiche intracellulari del Ca2+ nelle singole cellule con esperimenti di Ca2+-imaging in fettine precedentemente incubate con la sonda fluorescente FURA2-AM. Ho analizzato piccoli gruppi di interneuroni localizzati nella regione ventrale del midollo spinale, a stadi sia precoci che tardivi di sviluppo della rete, cioè a 7-11 (prima settimana) e 14-17 (seconda settimana) giorni in vitro (DIV; Furlan et al., 2007). Per la prima volta ho descritto un cambiamento nella generazione di segnali spontanei al Ca2+, dipendente dalla maturazione in vitro delle colture: da waves precoci, guidate dall’attività sinaptica, che invadevano l’intera regione ventrale del midollo spinale, fino a tardive oscillazioni asincrone, indipendenti dall’attività elettrica, generate da pochi neuroni ristretti alle aree ventrali. Mediante marcature di immunofluorescenza nonché con esperimenti di Ca2+- imaging, ho dimostrato che solo una minoranza (dal 15 al 20 %) di neuroni presenti nelle zone ventrali esprimevano questa tardiva attività oscillatoria. Queste oscillazioni mostravano una specifica dipendenza dalle proprietà di buffering del Ca2+ presenti a livello mitocondriale (Fabbro et al., 2007). In seguito, ho valutato il ruolo che le fonti extracellulari e intracellulari di Ca2+ potevano avere nella generazione di queste oscillazioni indipendenti dall’attività elettrica. Una prima idea del fatto che tali oscillazioni avessero un’origine complessa, Abstract 6 derivava dall’osservazione che nella maggior parte delle cellule (60%), questi segnali erano completamente bloccati in una soluzione priva di Ca2+, mentre nel 40% dei neuroni alcune oscillazioni persistevano anche in assenza di Ca2+. Questa risposta in una soluzione priva di Ca2+ è risultata essere bimodale, dal momento che non ho mai riscontrato alcuna coesistenza di questi due fenomeni nella stessa fettina. Una simile eterogeneità è stata osservata anche in seguito ad applicazioni di tapsigargina, la quale induceva sia il blocco (62% di neuroni) che la persistenza (38%) delle oscillazioni. Questa attività oscillatoria non dipendeva, però, dai depositi intracellulari di Ca2+ sensibili alla rianodina. Così, nonostante le proprietà stereotipate delle oscillazioni (origine, periodicità, etc…), questi eventi potrebbero essere generati grazie al contributo di diverse fonti di Ca2+. Una seconda questione importante nell’identificazione dei neuroni oscillanti è stata quella di monitorare i pattern di espressione delle Ca2+ binding proteins e dei trasportatori del Cl-, KCC2 e NKCC1. Ho osservato una forte dipendenza del profilo di espressione della proteina calbindina in relazione alla maturazione dei circuiti ventrali durante lo sviluppo. Questo non era, però, un fenomeno universale, infatti, altre Ca2+ binding proteins, come calretinina e parvalbumina, non avevano lo stesso profilo di espressione. Non ho, invece, riscontrato differenze nell’espressione della proteina NKCC1 tra 1 e 2 settimane in coltura; al contrario KCC2, andando avanti con lo sviluppo, si trovava maggiormente localizzata nei processi neuronali. Risultati recenti dimostrano che l’H2O2 è un donatore endogeno di specie reattive dell’ossigeno, presente nel CNS in concentrazioni μM (Lei et al., 1998). Nel midollo spinale post-natale l’H2O2 è stata recentemente indicata anche come un mediatore solubile dipendente dal Ca2+ intracellulare, capace di modulare la plasticità sinaptica in condizioni sia fisiologiche che patologiche (Takahashi et al., 2007). In questo mio studio, concentrazioni fisiologiche di H2O2 aumentavano il livello basale del Ca2+ intracellulare solo nei neuroni che oscillavano, senza però cambiare il periodo delle oscillazioni. Il fatto che i neuroni oscillanti fossero le sole cellule che rispondevano a basse dosi di H2O2 ci ha suggerito che questi interneuroni spinali potrebbero essere dei critici trasduttori dell’azione modulatoria dell’H2O2. In questo modo, un piccolo gruppo di interneuroni ventrali (a 2 settimane di crescita in vitro) potrebbe essere caratterizzato da due marcatori funzionali: la sensibilità all’ H2O2 e la capacità di produrre oscillazioni spontanee. Sembra molto interessante supporre che le periodiche oscillazioni al Ca2+ e la sensibilità all’H2O2 conferiscano a queste cellule la capacità di modellare la plasticità dei circuiti locali attraverso differenti cambiamenti (fasici o tonici) nella concentrazione del Ca2+ intracellulare.
The development of ventral spinal networks into functional circuits is a complex process comprising genetic and epigenetic mechanisms cooperating for the maturation of motor control (Jessell, 2000; Kiehn, 2006). Elucidating such a process is crucial in modern neuroscience to identify neurons more vulnerable to spinal degenerative disease or to develop novel strategies for rebuilding damaged circuits. Organotypic cultures developed from embryonic mouse spinal cord, maintained in vitro for 1 or 2 weeks, recapitulate many events of the in vivo developing spinal segments and are particularly suited to study spinal network maturation (Avossa et al., 2003; Rosato-Siri et al., 2004; Furlan et al., 2005; Furlan et al., 2007). In this thesis, I used such a model to investigate, in embryonic spinal segments, the spatio-temporal control of intracellular Ca2+ signaling generated by neuronal populations in motor circuits. I investigated the age-dependent expression of repetitive Ca2+ signals monitoring, by Ca2+-imaging technique, neuronal Ca2+ dynamics at single cell level in slice cultures of the embryonic mouse spinal cord, loaded with the fluorescent indicator FURA2-AM. I analyzed small groups of ventral spinal neurons at early and late embryonic network developmental stages, namely at 7-11 (1 week) and 14-17 (2 weeks) days in vitro (DIV; Furlan et al., 2007). I reported, for the first time, the developmentally-regulated shift in the generation of repetitive Ca2+ signals, from early waves driven by synaptic activity invading the entire spinal region to late, activity-independent, asynchronous oscillations generated by few neurons in restricted ventral areas. I demonstrated by immunofluorescence stainings and Ca2+-imaging experiments, that only a minority (15 to 20 %) of ventral neurons expressed this late Ca2+ oscillatory activity. Such oscillations expressed a specific dependence on mitochondria Ca2+ buffering properties (Fabbro et al., 2007). Next, I addressed the role of the extracellular and intracellular Ca2+ sources in the generation of activity independent oscillations. A first glimpse about the complex origin of Ca2+ for oscillations came from the observation that, in the majority of cells (60%), oscillations were completely abolished by Ca2+-free solution, whereas in 40% of cells clusters of oscillations were still detected during Ca2+-free perfusion. This response to Ca2+-free medium was bimodal, as no coexistence of these two effects was found in the same slice. Similar heterogeneity was observed following the application of the Ca2+ stores depletory, thapsigargin that induced either block (62% of neurons) or persistence (38%) of oscillations. The oscillatory activity was not dependent on ryanodine-sensitive stores. Abstract 4 Thus, despite the stereotyped properties of oscillations (origin, periodicity, etc), these events could be generated with the contribution of multiple Ca2+ sources. A second issue relevant in identifying oscillating neurons was to monitor the patterns of expression of Ca2+ binding proteins and of Cl- co-transporters, KCC2 and NKCC1. I observed a strong dependence of the expression profile of the Ca2+-binding protein calbindin on developmental maturation. This was not an universal phenomenon, in fact, other Ca2+ binding proteins, such as calretinin and parvalbumin, did not follow the same pattern. I did not detect differences in the expression pattern of NKCC1, between 1 and 2 weeks of in vitro growth, conversely KCC2-ir was more located to neuronal processes along with development. Recent results show that H2O2 is an endogenous donor of reactive oxygen species present in the CNS in μM concentrations (Lei et al., 1998). In the postnatal spinal cord, H2O2 has been recently indicated as a soluble, Ca2+ dependent mediator, capable of modulating synaptic plasticity under physiological and pathological conditions (Takahashi et al., 2007). In this study, physiological concentrations of H2O2 increased intracellular Ca2+ only in oscillating neurons without changing the oscillation period. The fact that oscillating neurons were the only responsive cells to a low H2O2 dose suggested that these spinal interneurons could be critical transducers of the modulatory action of H2O2. Thus, a small group of ventral interneurons (at 2 weeks in vitro) could be characterized by two functional predictors, namely sensitivity to H2O2 and ability to produce spontaneous oscillations. It seems attractive to assume that periodic oscillations of Ca2+ plus H2O2 sensitivity confer a summative ability to these cells to shape the plasticity of local circuits through different changes (phasic or tonic) in intracellular Ca2+.
XXI Ciclo
1980

2011/2012
Regenerative medicine is a broad interdisciplinary field, tremendously grown in the last decades, which encompasses several different research areas, such as biomaterial sciences and tissue engineering, whose unifying concept holds an enormous therapeutic potential, being that of restoring impaired organs or tissue functions, resulting from congenital defects, trauma or disease (Greenwood et al.; 2006; Mason and Dunnill, 2008). The main challenge faced by tissue engineering is the need to have a renewable source of cells and biomaterials possessing the right mechanical, chemical and biological features, to create constructs resembling native tissues. The design of scaffolds able to support and promote tissue regeneration and/or functional restore, in particular, is a critical step for the success of an implant, as it should recapitulate the complex architecture of the physiological microenvironment, e.g. the appropriate extracellular matrix, which has been shown to actively direct the behaviour of cells, through both chemical and physical cues (Daley et al., 2008; Place et al., 2009; Rozario and DeSimone, 2010). In this context, nanotechnology tools may greatly enhance the success of tissue engineering strategies, by providing the chance of producing surfaces and materials with topographical features that mimic the natural ones, in addition to the possibility to functionalize nanomaterials with bioactive molecules (Gelain et al., 2008; Zhang and Webster , 2009; Dvir et al., 2011; Koh et al., 2008). Among nanomaterials, carbon nanotubes (CNTs) stood out, since their discovery, for their outstanding mechanical, electrical and thermal properties, like their extraordinary strength coupled with remarkable flexibility, or their high electrical conductivity, which make them a well-suited platform technology for biomedical applications. Recently, several works have been published, which support the use of CNT-based scaffolds to promote neuronal attachment, differentiation and growth (Mattson et al., 2000; Hu et al., 2004; Hu et al., 2005; Galvan-Garcia et al., 2007). Moreover, in the last decade, our group showed that CNT/neuronal hybrid networks show a boost in synaptic transmission (Lovat et al., 2005; Mazzatenta et al., 2007) and that the direct contact established between single CNT and neuronal membranes affect single neuron integrative abilities (Cellot et al., 2009), besides promoting network connectivity and synaptic plasticity phenomena in cortical cultured circuits (Cellot et al., 2011). Here, to extend our knowledge about interactions between CNT and neurons, we long-term cultured organotypic spinal explants, possessing a complex multilayered cytoarchitecture, with highly purified MWCNT scaffolds and then investigated, via a multidisciplinary approach, their growth and synaptic activity. Our aim was to verify whether and how a CNT-induced effect on neuronal performance could be transferred to network locations, which are far from the neuronal/MWCNT layer of interaction, but sinaptically communicating with it. We documented, via TEM investigations, the presence of tight connections established between the neuronal membranes of neurons belonging to the bottom layer of the spinal tissue and the CNT meshwork underlying it. By means of confocal microscopy, SEM and AFM techniques, we showed, for the first time, that the long-term interfacing of spinal cord explants to CNTs induced an increase in the number and length of peripheral neuronal fibres outgrowing the spinal tissue, associated to changes in growth cone activity and in fibre elastomechanical features. We also demonstrated, via patch-clamp recordings performed from interneurons in the ventral (premotor) area of the explants, that both spontaneous and evoked synaptic currents displayed a potentiation in the presence of the CNT scaffold, detected as an increase in current amplitude in neurons which were as far as 5 cell layers from the tissue/substrate site of interaction. We speculate that these two effects (the increased fibres growth and the boosting in synaptic activity) rely upon two different mechanisms, a direct and a remote one, by which CNTs affect the spinal tissue development. Indeed, the first exerted on fibres directly adhering to the CNT substrate, while the second is likely to be mediated by alterations occurring at the tissue layer integrated with CNTs, which are transmitted, through a remote effect, to distant network locations, synaptically communicating with such a layer. These results support the hypothesis that CNTs may be employed to boost spinal neurite re-growth and functional spinal performance, in the perspective of re-establishing the physical and functional communication between disconnected spinal segments, We therefore decided to implement a model in which two organotypic spinal explants grow together on the same support, as a useful model for neuronal reconnection investigations and to test the possibility that a CNT-based scaffold, interposed between the two explants, may act as a bridge to promote the physical and electrical communication between the two spinal segments. By means of immunostaining experiments and confocal microscopy we reported the presence of a huge amount of fibres, projecting from the two spinal slices and integrating in a complex network, especially localized in the DRG regions, while very few fibres seemed to directly connect the two explanted tissues at the level of the explants cores. When, via voltage-clamp pair recordings, we looked for the presence of an electrical reconnection between explants, we found a small percentage of co-cultured explants displaying a complex coupled behaviour, detected as a strongly correlated bursting activity. These preliminary data seem to confirm the goodness of such an in vitro model to investigate the intrinsic reparative potential of spinal cord tissue and to improve such ability via nanotechnological tools.
XXV Ciclo
1982

Spinal cord injury (SCI) is characterized to be a two-step process composed by the primary lesion consisting of the initial trauma and the secondary damage, characterized by multiple processes including inflammation, oxidative stress and cell death that lead to a significant expansion of the original damage and to an increase of the functional deficit. Among the aforementioned processes, the oxidative stress plays a significant role in pathophysiology of SCI. In this study, we evaluated the role of melatonin, potent antioxidant and immunomodulator indoleamin, on the oxidative stress, the tissue viability and the neuritic plasticity deriving from the gray matter in an experimental model of organotypic cultures. These cultures consisted of Sprague Dawley rat spinal cord slice treated with hydrogen peroxide (H2O2). In five experimental groups, A) Control Group (CTR) – Organotypic spinal cord slice culture (350μm); B) Stressed Group (H2O2) – Organotypic spinal cord slice culture (350μm) exposed to H2O2 (50 μM); C) Control Group treated with melatonin (10-5M) of 24 hours (CTR+MEL) – Organotypic spinal cord slice culture (350μm) treated with melatonin for 24 hours; D) Treated Group (H2O2+MEL-POST) – Organotypic spinal cord slice culture (350μm) exposed to H2O2 (50 μM) and treated after 24 hours with melatonin (10-5M) for 24 hours; E) Treated Group (H2O2+MEL-PRE) – Organotypic spinal cord slice culture (350μm) pre-treated with melatonin (10-5M) for 24 hours (50 μM) and exposed to H2O2 for other 24 hours. We investigated the slice cellular death by propidium iodide (PI) assay, the slice vitality by MTT assay, the superoxide dismutase (SOD) and total thiols (SH) levels for the contrast to the oxidative stress, the neuronal (NeuN) and the synaptophysin (Syp) immunopositivity. Melatonin significantly decreased the number of dead cells, increased slice vitality, mainly in slices treated before H2O2 exposition. Melatonin enhanced SOD immunopositivity, contrasted total thiols decrease, attenuated Syp reduction and increased NeuN immunopositivity. Overall, these findings suggest that melatonin may exert a potentially beneficial effect upon the progression of SCI secondary damage, protecting the tissue from a further degeneration.

Tous les réseaux de neurones immatures génèrent une activité dite « spontanée »qui persiste même en l’absence de toute afférence et est impliquée dans de nombreux processus développementaux. Cette activité apparaît in vitro sous formes de vagues calciques ou électriques pouvant se propager sur de grandes distances et embraser toute la préparation. Toutefois, sa dynamique a été assez peu étudiée jusqu’à présent. En vue de combler quelque peu cette lacune, nous avons utilisé des matrices de microélectrodes (MEA) pour caractériser l’activité rythmique spontanée dans la moelle épinière embryonnaire de souris, sur des préparations aigues et en culture incluant également le tronc cérébral.Les enregistrements MEA produisent des volumes de données très importants qui nécessitent des outils d’analyse performants et adaptés à l’information que l’on souhaite extraire. Nous avons donc développé des méthodes pour la détection, la classification et la cartographie des patrons spatiotemporels d’activité dans les données multicanaux. Notre approche cartographique utilise l’interpolation par splines et est orientée vers la production de cartes multimodales combinant l’activité électrique et des données anatomiques ou biochimiques (marquages). Ces méthodes d’analyse nous ont permis de décrire très précisément l’évolution de l’activité spontanée aux stades précoces (E12.5–E15.5). Nous avons également montré que, à E14.5, l’activité est initiée dans le bulbe, plus précisément dans une région riche en neurones 5-HT, suggérant un nouveau rôle des voies sérotoninergiques descendantes dans la maturation des réseaux spinaux.Enfin, nous avons analysé les mouvements embryonnaires à E14.5 et avons découvert des caractéristiques analogues à la dynamiques spatiotemporelle des activités intraspinales
Immature neural networks generate a peculiar type of activity that persists even in the absence of electrical inputs and was termed for this reason “endogenous”or “spontaneous”. This activity is ubiquitous and was found involved in a wide range of developmental events. In vitro, it can be observed as calcium or electrical waves propagating over great distances, often invading the whole preparation,but its dynamics remain poorly described. In order to somewhat fill this gap,we used multielectrode arrays (MEAs) to characterise the spontaneous rhythmic activity in the mouse developing spinal cord, in both acute and cultured isolated hindbrain-spinal cord preparations.To extract relevant information from the massive amounts of data yielded by MEA recordings, adapted analysis tools are needed. Thus, we have developedmethods for the detection, classification and mapping of spatiotemporal patternsof activity in multichannel data. Our mapping approach is based on the thin plates pline interpolation and includes the possibility to combine maps of activity with anatomical or stained data for multimodal imaging.These methods allowed us to analyse in great detail the evolution of spontaneousactivity at early stages (E12.5–E15.5). In addition, we have localised theinitiation site of E14.5 activity in the medulla and shown that it matches a densemidline population of serotoninergic neurons, suggesting a new role for 5-HTpathways in the maturation of spinal networks. Finally, we have recorded andtracked spontaneous limb movements of E14.5 embryos and found that features of motility were consistent with patterns of spinal activity

Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease characterized by the loss of upper and lower motor neurons (MNs) and glia dysfunction, which con-tribute to disease onset and progression. miRNA dysregulation and neuroinflammation have been pointed to contribute for MN degeneration in ALS. Lately, our group demon-strated that miR-124 was increased in SOD1G93A (mSOD1) NSC-34 MNs and in their de-rived sEVs, causing an early microglia activation followed by cell repairing mechanisms. Increased expression of miR-124 was also identified in the spinal cord (SC) of mSOD1 mice during the symptomatic stage, indicating a potential pathological role of this miRNA in ALS. Interestingly, the secretome from mSOD1 MNs transfected with anti-miR-124 was promising in reverting the damage-associated mechanisms since it prevents neurodegeneration, microglia activation and the pathology associated to neuro-immune homeostatic imbalance in spinal cord organotypic cultures (SCOCs) from mSOD1 mice. Moreover, unpublished studies from our group have shown increased levels of miR-125b in micro-glia isolated from the SC of mSOD1 mouse pups and cultured for 16 days in vitro (aged cells). miR-125b was also found increased in mSOD1 NSC-34 MNs, while it was shown to be upregulated in the SC of symptomatic/end-stage mSOD1 mice and to drive microglia activation and MN death in ALS. Here, our major goal was to explore the potential therapeutic effects of secretomes from MNs and microglia after reparative strategies with anti-miR-124 and anti-miR-125b towards translation into clinics. Our results highlight that secretome from mSOD1 MNs with normalized levels of miR-124 and miR-125b counter-acted some abnormalities found in the mSOD1 SCOCs such as axonal transport, phagocytic ability and oligodendrocytes maturation. Our data also suggest that the combined usage of anti-miR-124 and anti-miR-125b modulatory strategies may provide a better therapeutic approach to be adopted. Further, although both secretomes from either modulated mSOD1 MNs or mSOD1 microglia are able to restore cellular homeostasis in mSOD1 SCOCs, our findings suggest that modulated mSOD1 MNs secretome produces more benefits in comparison with those from modulated mSOD1 microglia in ALS, for early stages of disease progression.
A Esclerose Lateral Amiotrófica (ELA) é uma doença neurodegenerativa caracteri-zada pela perda de neurónios motores (NMs), superiores e inferiores, e pela disfunção da glia, que contribuem para o início e progressão da doença. A desregulação de miRNAs e a neuroinflamação têm sido apontados como fatores que contribuem para a degeneração dos NMs na ELA. Recentemente, o nosso grupo encontrou níveis aumentados de miR-124 em NMs SOD1G93A (mSOD1) NSC-34 e em pequenas vesículas extracelulares libertadas por estes NMs mutados, o que resultou numa ativação precoce da microglia, seguida de mecanismos de reparação celular. O aumento da expressão de miR-124 também foi identificado na medula espinhal de ratinhos mSOD1 durante a fase sintomática da doença, indicando um potencial papel patológico deste miR-124 na ELA. Curiosamente, o secretoma de NMs mSOD1 transfectados com anti-miR-124 demonstrou ser promissor na reversão de mecanismos danosos, uma vez que preveniu a neurodegeneração, ativação da microglia e o desequilíbrio neuroimune associado à patologia, em culturas organotípicas de medula espinhal de ratinhos mSOD1. Para além disso, estudos não publicados do nosso grupo mostraram níveis aumentados de miR-125b em microglia isolada da medula espinhal de ratinhos recém-nascidos mSOD1 e em microglia em cultura por 16 dias in vitro (células envelhecidas). miR-125b também foi encontrado aumentado em NMs mSOD1 NSC-34 (dados não publicados) e na medula espinhal de ratinhos mSOD1 sintomáticos/em fase final, o que conduziu à ativação microglial e à morte de NMs na ELA. Neste trabalho, o principal objetivo foi explorar os potenciais efeitos terapêuticos dos secretomas de NMs mSOD1 e de microglia mSOD1 após a aplicação de estratégias reparativas com anti-miR-124 e anti-miR-125b, usando culturas organotípicas (COs) de medula espinhal de ratinho, de modo à aplicação futura da terapia num contexto clínico. Os nossos resultados realçam que o secretoma de NMs mSOD1 com níveis normalizados de miR-124 ou miR-125b tem a capacidade de neutralizar algumas anomalias encontradas em mSOD1 de COs de medula espinhal, como o transporte axonal, capacidade fagocítica e maturação de oligodendrócitos. Os nossos dados revelam também que o uso combinado de ambas as estratégias modulatórias, de anti-miR-124 e anti-miR-125b, pode corresponder à melhor abordagem terapêutica a ser adotada. Por outro lado, embora ambos os secretomas de NMs mSOD1 e microglia mSOD1 sejam capazes de restaurar a homeostase celular em mSOD1 de COs de medula espinhal, os nossos resultados sugerem que o secretoma de NMs mSOD1 modula-dos parece produzir mais efeitos benéficos em comparação com os produzidos pelo secretoma de microglia mSOD1 modulada em ALS, nesta fase inicial da progressão da doença.

Amyotrophic Lateral Sclerosis (ALS) is a fatal disease characterized by the degeneration of upper (cortical) and lower (spinal cord, SC) motor neurons (MNs) and aberrancy of glial cells. Results from our group point to a close connection between increased levels of miRNA-124 and the acquisition of pathological characteristics in MNs, astrocytes and microglia in ALS. Our main aim was to validate if the downregulation of the elevated levels of miR-124 in hSOD1G93A (mSOD1) MNs toward normal levels was preventive over neurodegeneration, astrocyte aberrancies and microglia activation in the mSOD1 mice at the early onset of the disease (10-12 weeks). Two ALS models were used: the NSC-34 MN-like cell line expressing mSOD1 (transgenic, TG) or not (wild-type, WT); and the SC organotypic cultures (OCs) from WT and TG mice. Pathological differences between TG and WT SCOCs were investigated. Relatively to the MN models, we used the modulation with pre-miR-124 (only in WT) and that of anti-miR-124 (only in the TG). The isolated secretomes were incubated in WT and TG SCOCs to assess harmful and/or neuroprotective properties. In TG SCOCs we observed: (i) increased necrotic cell death; (ii) disturbed inflammatory-associated miRNAs (increase in miR-21/miR-146a); (iii) and dysregulated neuronal and glial genes (increased CX3CR1, IL-1β, IL-10, SYP, DRP1, GLT-1 and downregulation of iNOS, HMGB1, Dlg4, CX3CL1 and GFAP). WT-MN secretome counteracted pathological markers in TG SCOCs. In contrast, TG MN secretome induced deleterious effects in WT SCOCs. Secretome from miR-124-enriched WT MNs incubated in WT SCOCs led to a profile of miRNAs and protein-coding genes similar to that caused by the TG MN secretome. On the contrary, the secretome from TG MNs depleted in miR-124 restored a deactivated profile in TG SCOCs. Our data reveals MN upregulation of miR-124 as a key player in ALS pathological processes.
Casa da Misericórdia de Lisboa (SCML), project ref. ALSResearch Grant ELA-2015-002