Heating represents a promising approach to induce neurite outgrowth and neuronal function recovery. In previous studies, protocols with different temperatures (from 38°C to 50°C) and durations (from milliseconds to several days) could induced differentiation of different cell types like Xenopus laevis oocytes, cultured mammalian cells, neurons, stem cells and cancer cells. This effect has been attributed to changes in cell membrane capacitance and in ion channel properties, but the underlying mechanism remains so far unknown. The present project was aimed to investigate the eventual modifications, induced by two approaches of thermal stimulation, on the behaviour of a model of dorsal root ganglion (DRG) neurons, the F-11 cell line, previously characterized in our laboratory. These cells could express ion channels and cell membrane receptors consistent with those of sensory neurons and could be employed as a good model to study neuronal proliferation and differentiation mechanisms. Initially, to test if heating could effectively induce differentiation of our cellular model, we performed experiments of bulk stimulation: cells were placed in an incubator at different time and temperature combinations for two consecutive days. Thus, morphological and functional analysis were performed to investigate neuronal differentiation until 8 days. Results showed a significant difference in neurite elongation and in electrophysiological properties (resting membrane potential, Na+ and K+ current density and action potential frequency) in samples maintained at 41,5°C for 30 minutes versus 37°C samples. An intracellular Ca2+ signal analysis evoked by Capsaicin was performed to verify the involvement of TRPV (Transient Receptor Potential Vanilloid) channels in the effects of heating. Results showed that the Ca2+ signal was higher in heated cells compared to the control, suggesting that the treatment could increase the expression and/or the properties of TRPV1 channels, which are permeable to Ca2+. Moreover, we performed a lactate dehydrogenase activity (LDH) assay to verify if the treatment could induce cell stress and results showed that heat had no detrimental effects on F-11 cells. Considering these results, we investigated the effects of a scalable thermal stimulation method, established by irradiating Prussian Blue nanoparticles (PBNPs) with a near infrared laser. A disk of PBNPs-PVA was placed on the outer surface of the petri dish in which the cells were seeded, avoiding a direct contact between the material and the cells, and it was irradiated by a near infrared laser to increase culture medium temperature to 41,5°C. Neurite elongation was significantly increased in irradiated cells compared to non-irradiated control cells and significant differences were also observed during the functional analysis by patch-clamp technique. To verify if the effects on cellular properties could be maintained for a longer period, we performed a functional investigation also on heated and irradiated cells after 12 days in culture. Results showed that F-11 cells could maintain a differentiated phenotype also after 12 days in culture. The heating techniques used in literature could modify neuron excitability and had effects on cell morphology and staining, but the functional effects has not been reported so far. Moreover, primary cells represent an ideal model to study sensory neurons, but their availability is limited. The use of an immortalized cell line allowed to perform a functional study on the effects of heating, and the results demonstrated that a targeted thermal stimulation could be a promising approach to induce cell differentiation and support the future application of this method as a strategy to modify neuronal behaviour in vivo.
La stimolazione termica è una tecnica esplorata negli ultimi anni in quanto promettente per il differenziamento cellulare. Studi precedenti hanno dimostrato che questo metodo può indurre il differenziamento di diverse tipologie cellulari, dalle staminali alle tumorali, probabilmente attraverso cambiamenti della capacità della membrana e delle proprietà biofisiche dei canali ionici. Tuttavia, i meccanismi che sostengono questo processo restano a oggi sconosciuti. Il presente progetto ha l’obiettivo di studiare gli effetti della stimolazione termica sul comportamento di un modello in vitro di neuroni dei gangli delle radici dorsali (DRG), la linea cellulare F-11, caratterizzata nel nostro laboratorio. Queste cellule possono esprimere canali ionici e recettori di membrana, caratteristici dei neuroni sensoriali, e possono essere impiegate come modello per studiare i meccanismi coinvolti nella proliferazione e nel differenziamento neuronale. Inizialmente, per valutare gli effetti della tecnica di riscaldamento sul modello cellulare scelto, abbiamo effettuato esperimenti di stimolazione “in bulk” utilizzando un incubatore. Le cellule sono state poste nell’incubatore ed esposte a temperature diverse per intervalli di tempo differenti, per due giorni consecutivi. Analisi morfologiche e funzionali sono state condotte a partire dalla semina fino a 8 giorni. I risultati hanno mostrato una differenza significativa nella lunghezza dei neuriti e nelle proprietà elettrofisiologiche (potenziale di membrana, densità di corrente di Na+ e K+, e frequenza della scarica dei potenziali d’azione) nei campioni mantenuti a 41,5°C per 30 minuti rispetto al controllo (mantenuto a 37°C). È stata inoltre condotta un’analisi dei segnali intracellulari di Ca2+ indotti da capsaicina, per verificare l’eventuale coinvolgimento dei canali TRPV (Transient Receptor Potential Vanilloid) negli effetti indotti dal calore. I risultati hanno mostrato segnali intracellulari di Ca2+ maggiori nelle cellule riscaldate rispetto al controllo, suggerendo che l’esposizione al calore potrebbe influenzare l’espressione e/o le proprietà dei canali TRPV1, permeabili al Ca2+. Inoltre, il saggio della lattato-deidrogenasi (LDH) ha permesso di escludere effetti citotossici della metodica utilizzata sui campioni trattati. Considerando questi risultati, abbiamo studiato gli effetti di una stimolazione termica localizzata, ottenuta tramite l’irraggiamento di nanoparticelle Prussian Blue (PBNP) con un laser nel vicino infrarosso (NIR). Le PBNP sono state applicate sulla superficie esterna delle petri in cui sono state seminate le cellule, evitando in questo modo il contatto tra di esse e le cellule. Anche con questo approccio si sono registrate differenze significative nella lunghezza dei neuriti e nelle proprietà elettrofisiologiche, confermando l’induzione al differenziamento. Per verificare che l’effetto sulle proprietà cellulari potesse mantenersi nel tempo, ulteriori analisi delle proprietà morfologiche e funzionali sono state condotte a tempi successivi agli 8 giorni. I risultati hanno mostrato che le cellule F-11 possono mantenere un fenotipo differenziato anche a 12 giorni. Le tecniche di riscaldamento utilizzate in letteratura sono risultate efficaci per modificare l’eccitabilità dei neuroni e hanno effetti sulla morfologia, ma a oggi non sono stati ancora riportati gli effetti funzionali. Inoltre, le cellule primarie rappresentano un modello ideale per studiare i neuroni sensoriali, ma la loro disponibilità è limitata. L’uso di una linea immortalizzata ha permesso di effettuare uno studio funzionale degli effetti del riscaldamento, e i risultati ottenuti dimostrano che una stimolazione termica localizzata può essere un metodo promettente per indurre il differenziamento e supportano la possibile applicazione futura di questa tecnica come una nuova strategia per modificare il comportamento neuronale in vivo.
(2022). Differentiation of a dorsal root ganglion neuron model induced by a novel approach of thermal stimulation: a morphological and functional investigation. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2022).
Differentiation of a dorsal root ganglion neuron model induced by a novel approach of thermal stimulation: a morphological and functional investigation
BLASA, STEFANIA
2022
Abstract
Heating represents a promising approach to induce neurite outgrowth and neuronal function recovery. In previous studies, protocols with different temperatures (from 38°C to 50°C) and durations (from milliseconds to several days) could induced differentiation of different cell types like Xenopus laevis oocytes, cultured mammalian cells, neurons, stem cells and cancer cells. This effect has been attributed to changes in cell membrane capacitance and in ion channel properties, but the underlying mechanism remains so far unknown. The present project was aimed to investigate the eventual modifications, induced by two approaches of thermal stimulation, on the behaviour of a model of dorsal root ganglion (DRG) neurons, the F-11 cell line, previously characterized in our laboratory. These cells could express ion channels and cell membrane receptors consistent with those of sensory neurons and could be employed as a good model to study neuronal proliferation and differentiation mechanisms. Initially, to test if heating could effectively induce differentiation of our cellular model, we performed experiments of bulk stimulation: cells were placed in an incubator at different time and temperature combinations for two consecutive days. Thus, morphological and functional analysis were performed to investigate neuronal differentiation until 8 days. Results showed a significant difference in neurite elongation and in electrophysiological properties (resting membrane potential, Na+ and K+ current density and action potential frequency) in samples maintained at 41,5°C for 30 minutes versus 37°C samples. An intracellular Ca2+ signal analysis evoked by Capsaicin was performed to verify the involvement of TRPV (Transient Receptor Potential Vanilloid) channels in the effects of heating. Results showed that the Ca2+ signal was higher in heated cells compared to the control, suggesting that the treatment could increase the expression and/or the properties of TRPV1 channels, which are permeable to Ca2+. Moreover, we performed a lactate dehydrogenase activity (LDH) assay to verify if the treatment could induce cell stress and results showed that heat had no detrimental effects on F-11 cells. Considering these results, we investigated the effects of a scalable thermal stimulation method, established by irradiating Prussian Blue nanoparticles (PBNPs) with a near infrared laser. A disk of PBNPs-PVA was placed on the outer surface of the petri dish in which the cells were seeded, avoiding a direct contact between the material and the cells, and it was irradiated by a near infrared laser to increase culture medium temperature to 41,5°C. Neurite elongation was significantly increased in irradiated cells compared to non-irradiated control cells and significant differences were also observed during the functional analysis by patch-clamp technique. To verify if the effects on cellular properties could be maintained for a longer period, we performed a functional investigation also on heated and irradiated cells after 12 days in culture. Results showed that F-11 cells could maintain a differentiated phenotype also after 12 days in culture. The heating techniques used in literature could modify neuron excitability and had effects on cell morphology and staining, but the functional effects has not been reported so far. Moreover, primary cells represent an ideal model to study sensory neurons, but their availability is limited. The use of an immortalized cell line allowed to perform a functional study on the effects of heating, and the results demonstrated that a targeted thermal stimulation could be a promising approach to induce cell differentiation and support the future application of this method as a strategy to modify neuronal behaviour in vivo.File | Dimensione | Formato | |
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Descrizione: Differentiation of a dorsal root ganglion neuron model induced by a novel approach of thermal stimulation: a morphological and functional investigation
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Doctoral thesis
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