Background: Neurosensory degeneration of the ear refers to the progressive deterioration of the sensory structures responsible for hearing within the cochlea, including damage to hair cells and their neural connections. This can lead to hearing loss and disorders. Hair cells convert sound waves into neural signals. Factors such as genetics, aging, noise, drugs, and infections cause damage. Promoting neurite growth is crucial for the nervous system and auditory disorders. In vitro acoustic stimulation provides a controlled environment to promote neural growth. Aim: The research objectives concern the establishment of an in vitro acoustic stimulation system to promote neurite growth, and include setting up the system, investigating the effects of acoustic stimulation on neurite growth, comparing growth promotion in healthy and damaged cells, exploring the impact on neural and sensory cell, and evaluating effects at different frequencies. The research hypotheses include that acoustic stimulation will enhance neurite growth, effects could vary among cell types, stimulation could promote growth even in damaged cells, and the in vitro acoustic stimulation system could represent a promising therapeutic approach to enhance neural regeneration. Materials and Methods: The experimental setup of this project includes a custom acoustic exposure system housed in a specially designed incubator to achieve a controlled acoustic environment for cell cultures. The utilized cell cultures are PC12 and OCK3 cells, employed under three different experimental conditions: untreated/healthy (Nt), damaged with cisplatin at a concentration of 5 micromolar (Cpt5), and damaged with cisplatin at a concentration of 13 micromolar (Cpt13). They underwent 1 cycle and 3 cycles of controlled sound stimulation, each lasting 20 minutes, using the custom acoustic system. The cells were stimulated with specific frequency bands ranging from 500 Hz to 8000 Hz. Results were obtained through morphological analysis for the PC12 cell line and through a cell viability test for the OCK3 cell line. II Results: The study demonstrates that using specific sub-bands within the 500-4000 Hz frequency range is more effective in promoting neurite growth and regeneration compared to the entire range. Particularly, the sub-bands 500-1000 Hz, 1000-2000 Hz, and 2000-4000 Hz consistently show positive effects on damaged cells, suggesting their suitability for regenerative responses. Among these, the 2000-4000 Hz frequency range consistently displays the most significant positive outcomes. However, the broader 4000-8000 Hz frequency band does not consistently yield notable regenerative effects. This indicates the importance of tailored acoustic stimulation for specific frequency ranges to enhance neurite regeneration in damaged cells. Conclusion: In conclusion, the acoustic stimulation system holds potential for promoting neurite growth. Positive effects, including increased neurite length and number, indicate its benefits for damaged neurons under specific conditions. Applying acoustic stimulation within certain frequency ranges, especially between 2000 and 4000 Hz, consistently enhances neurite growth and regeneration. Moreover, comparing 1 cycle to 3 cycles of sound stimulation reveals repeated stimulation yields even more significant positive effects on neurite growth, implying cumulative impact. Based on these results, it can be inferred that acoustic stimulation fosters neurite growth in specific circumstances. While providing valuable insights, the study highlights the importance of frequency range selection for optimal regenerative outcomes. However, due to result heterogeneity and complexity, further studies are required to attain clarity and potentially elucidate mechanisms.
Background: La degenerazione neurosensoriale dell'orecchio si riferisce al deterioramento progressivo delle strutture sensoriali responsabili dell'udito all'interno della coclea, inclusi danni alle cellule ciliate e alle loro connessioni neurali. Ciò può portare a perdita dell'udito e disturbi uditivi. Le cellule ciliate convertono le onde sonore in segnali neurali. Fattori come la genetica, l'invecchiamento, il rumore, i farmaci e le infezioni causano danni. Promuovere la crescita delle neuriti è cruciale per il sistema nervoso e i disturbi uditivi. La stimolazione acustica in vitro fornisce un ambiente controllato per favorire la crescita neurale. Obiettivo: Gli obiettivi della ricerca riguardano la creazione di un sistema di stimolazione acustica in vitro per promuovere la crescita dei neuriti e includono la configurazione del sistema, l'indagine sugli effetti della stimolazione acustica sulla crescita dei neuriti, la comparazione della promozione della crescita neurale nelle cellule sane e danneggiate, l'esplorazione dell'impatto sulle cellule neurali e sensoriali e la valutazione degli effetti a diverse frequenze. Le ipotesi di ricerca includono che la stimolazione acustica aumenterà la crescita dei neuriti, che gli effetti potrebbero variare tra i tipi di cellule, che la stimolazione potrebbe promuovere la crescita anche nelle cellule danneggiate e che il sistema di stimolazione acustica in vitro potrebbe rappresentare un approccio terapeutico promettente per potenziare la rigenerazione neurale. Materiali e metodi: L'apparecchiatura sperimentale di questo progetto include un sistema personalizzato di esposizione acustica alloggiato in un incubatore appositamente progettato per ottenere un ambiente acustico controllato per le colture cellulari. Le colture cellulari utilizzate sono cellule PC12 e OCK3, impiegate in tre diverse condizioni sperimentali: non trattate/sane (Nt), danneggiate con cisplatino a una concentrazione di 5 micro-moli (Cpt5) e danneggiate con cisplatino a una concentrazione di 13 micro-moli (Cpt13). Sono state sottoposte a 1 ciclo e 3 cicli di stimolazione sonora controllata, ciascuno della durata di 20 minuti, utilizzando il sistema acustico personalizzato. Le cellule sono state stimolate con bande di frequenza specifiche comprese tra 500 Hz e IV 8000 Hz. I risultati sono stati ottenuti attraverso l'analisi morfologica per la linea cellulare PC12 e attraverso un test di vitalità cellulare per la linea cellulare OCK3. Risultati: Lo studio dimostra che l'utilizzo di specifiche sottobande all'interno della gamma di frequenza 500-4000 Hz è più efficace nel promuovere la crescita e la rigenerazione delle neuriti rispetto all'intera gamma. In particolare, le sottobande 500-1000 Hz, 1000-2000 Hz e 2000-4000 Hz mostrano in modo coerente effetti positivi sulle cellule danneggiate, suggerendo la loro idoneità per risposte rigenerative. Tra queste, la gamma di frequenza 2000-4000 Hz mostra in modo costante i risultati più significativamente positivi. Conclusioni: In conclusione, il sistema di stimolazione acustica ha il potenziale per promuovere la crescita dei neuriti. Gli effetti positivi, tra cui l'aumento della lunghezza e del numero, indicano i benefici per i neuroni danneggiati in condizioni specifiche. L'applicazione della stimolazione acustica all'interno di determinate gamme di frequenza, specialmente tra 2000 e 4000 Hz, migliora in modo coerente la crescita e la rigenerazione dei neuriti. Inoltre, il confronto tra 1 ciclo e 3 cicli di stimolazione sonora rivela che la stimolazione ripetuta produce effetti positivi ancora più significativi sulla crescita dei neuriti, implicando un impatto cumulativo.
Messa a punto di un sistema di stimolazione acustica in vitro al fine di promuovere la crescita dei neuriti
POLI, MARTINA
2022/2023
Abstract
Background: Neurosensory degeneration of the ear refers to the progressive deterioration of the sensory structures responsible for hearing within the cochlea, including damage to hair cells and their neural connections. This can lead to hearing loss and disorders. Hair cells convert sound waves into neural signals. Factors such as genetics, aging, noise, drugs, and infections cause damage. Promoting neurite growth is crucial for the nervous system and auditory disorders. In vitro acoustic stimulation provides a controlled environment to promote neural growth. Aim: The research objectives concern the establishment of an in vitro acoustic stimulation system to promote neurite growth, and include setting up the system, investigating the effects of acoustic stimulation on neurite growth, comparing growth promotion in healthy and damaged cells, exploring the impact on neural and sensory cell, and evaluating effects at different frequencies. The research hypotheses include that acoustic stimulation will enhance neurite growth, effects could vary among cell types, stimulation could promote growth even in damaged cells, and the in vitro acoustic stimulation system could represent a promising therapeutic approach to enhance neural regeneration. Materials and Methods: The experimental setup of this project includes a custom acoustic exposure system housed in a specially designed incubator to achieve a controlled acoustic environment for cell cultures. The utilized cell cultures are PC12 and OCK3 cells, employed under three different experimental conditions: untreated/healthy (Nt), damaged with cisplatin at a concentration of 5 micromolar (Cpt5), and damaged with cisplatin at a concentration of 13 micromolar (Cpt13). They underwent 1 cycle and 3 cycles of controlled sound stimulation, each lasting 20 minutes, using the custom acoustic system. The cells were stimulated with specific frequency bands ranging from 500 Hz to 8000 Hz. Results were obtained through morphological analysis for the PC12 cell line and through a cell viability test for the OCK3 cell line. II Results: The study demonstrates that using specific sub-bands within the 500-4000 Hz frequency range is more effective in promoting neurite growth and regeneration compared to the entire range. Particularly, the sub-bands 500-1000 Hz, 1000-2000 Hz, and 2000-4000 Hz consistently show positive effects on damaged cells, suggesting their suitability for regenerative responses. Among these, the 2000-4000 Hz frequency range consistently displays the most significant positive outcomes. However, the broader 4000-8000 Hz frequency band does not consistently yield notable regenerative effects. This indicates the importance of tailored acoustic stimulation for specific frequency ranges to enhance neurite regeneration in damaged cells. Conclusion: In conclusion, the acoustic stimulation system holds potential for promoting neurite growth. Positive effects, including increased neurite length and number, indicate its benefits for damaged neurons under specific conditions. Applying acoustic stimulation within certain frequency ranges, especially between 2000 and 4000 Hz, consistently enhances neurite growth and regeneration. Moreover, comparing 1 cycle to 3 cycles of sound stimulation reveals repeated stimulation yields even more significant positive effects on neurite growth, implying cumulative impact. Based on these results, it can be inferred that acoustic stimulation fosters neurite growth in specific circumstances. While providing valuable insights, the study highlights the importance of frequency range selection for optimal regenerative outcomes. However, due to result heterogeneity and complexity, further studies are required to attain clarity and potentially elucidate mechanisms.| File | Dimensione | Formato | |
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https://hdl.handle.net/20.500.12608/50912