Temporal lobe epilepsy (TLE) is one of the most frequent types of focal epilepsy in adulthood and it has been estimated that affects around 20% of all epileptic patients, representing a burden from the social, health and economic perspective. Several problems related to the pharmacological treatment and surgical resection of the epileptic focus in TLE make stem cell therapy an intriguing perspective. In order to restore the damaged neuronal circuits, Hermes project proposes an innovative strategy based on the combination of bioengineered brain tissue and neuromorphic microelectronics, supervised by artificial intelligence. This thesis focuses on the first steps of the project with the aim of fine-tuning the in vivo injection of the biological component into the ventral Cornus Ammonis 3, an area particularly damaged in TLE. We used the pilocarpine model of TLE in which the status epilepticus induced chemically in young adult rats leads to a chronic epileptic condition within 4 weeks from the treatment. Ibotenic acid was used to eliminate epileptic cells in the area where the biohybrid will self-form, avoiding contamination of the graft cells with the pathological ones. The volume of ibotenic acid to be injected was adjusted in order to guarantee a well-localized lesion in the desired area. Neural stem cells (NSCs), provided by our collaborators at University of Verona, were injected alone or with alginate, a bioactive extracellular matrix, provided by our partners at Italian Institute of Technology (IIT) in Genova, that should support the cells. The volume of alginate injected was chosen in order to optimize both the coverage of the injured area and the adequate support to the NSCs injected. The immunostaining analysis on microglia and astrocytes confirms the anti-inflammatory activity of alginate even in a very hostile context such as the injured epileptic brain. Our immunofluorescence data show that 9 weeks after transplantation cells are still alive and show an ongoing neuronal maturation process. These results offer important premises to achieve the goal of the project, supporting the possibility of reconstructing a functional neuronal circuit and implementing regenerative medicine with microelectronics and artificial intelligence.
L’epilessia del lobo temporale (TLE) è uno dei tipi più frequenti di epilessia parziale negli adulti ed è stato stimato che colpisce circa il 20% dei pazienti epilettici, rappresentando un onere dal punto di vista sociale, sanitario ed economico. Diversi problemi legati al trattamento farmacologico e alla resezione chirurgica del focus epilettico nella TLE rendono la terapia con cellule staminali una prospettiva sicuramente interessante. Per ripristinare i circuiti neuronali danneggiati, il progetto Hermes propone una strategia innovativa basata sulla combinazione del tessuto cerebrale bioingegnerizzato e microelettronica neuromorfica, supervisionata dall’intelligenza artificiale. Questa tesi si focalizza sui primi step del progetto con l’obiettivo di mettere a punto l’iniezione in vivo della componente biologica nell’ippocampo ventrale, in particolare nell’area del Corno d’Ammone 3 che è particolarmente danneggiata nella TLE. Abbiamo utilizzato il modello della TLE definito “pilocarpina” in cui lo stato epilettico indotto chimicamente mediante iniezione intraperitoneale di pilocarpina nei ratti giovani adulti porta a una condizione epilettica cronica entro 4 settimane dal trattamento. La successiva iniezione intracerebrale di acido ibotenico ha permesso di eliminare le cellule epilettiche nell’area ippocampale in cui si posizionerà il bioibrido, evitando quindi la contaminazione delle cellule iniettate con quelle patologiche. Il volume di acido ibotenico da iniettare è stato regolato in modo da garantire una lesione ben localizzata nell’area desiderata. Le cellule staminali neurali (NSCs), fornite dai nostri collaboratori dell’Università di Verona, sono state iniettate da sole oppure co-somministrate con alginato, una matrice extracellulare bioattiva, fornita dai colleghi dell’Istituto Italiano di Tecnologia (IIT) di Genova, che dovrebbe fungere da supporto per le cellule. Il volume di alginato iniettato è stato scelto in modo da ottimizzare sia la copertura dell’area lesionata che l’adeguato supporto alle NSCs iniettate. L’analisi di immunocolorazione specifica per microglia e astrociti ha confermato che l’alginato esercita anche un’attività anti-infiammatoria pur in presenza di un contesto molto ostile come il cervello epilettico lesionato. I dati di immunofluorescenza hanno dimostrato, 9 settimane dopo il trapianto, la sopravvivenza delle cellule trapiantate e la presenza di un processo di maturazione neuronale in corso. Questi risultati offrono premesse importanti per raggiungere l’obiettivo del progetto, supportando la possibilità di ricostruire un circuito neuronale funzionante e di implementare la medicina rigenerativa con la microelettronica e l’intelligenza artificiale.
Trapianto di cellule staminali neurali in un modello di ratto di epilessia del lobo temporale: un passo verso la medicina rigenerativa potenziata
RAIMONDI, FEDERICA
2020/2021
Abstract
Temporal lobe epilepsy (TLE) is one of the most frequent types of focal epilepsy in adulthood and it has been estimated that affects around 20% of all epileptic patients, representing a burden from the social, health and economic perspective. Several problems related to the pharmacological treatment and surgical resection of the epileptic focus in TLE make stem cell therapy an intriguing perspective. In order to restore the damaged neuronal circuits, Hermes project proposes an innovative strategy based on the combination of bioengineered brain tissue and neuromorphic microelectronics, supervised by artificial intelligence. This thesis focuses on the first steps of the project with the aim of fine-tuning the in vivo injection of the biological component into the ventral Cornus Ammonis 3, an area particularly damaged in TLE. We used the pilocarpine model of TLE in which the status epilepticus induced chemically in young adult rats leads to a chronic epileptic condition within 4 weeks from the treatment. Ibotenic acid was used to eliminate epileptic cells in the area where the biohybrid will self-form, avoiding contamination of the graft cells with the pathological ones. The volume of ibotenic acid to be injected was adjusted in order to guarantee a well-localized lesion in the desired area. Neural stem cells (NSCs), provided by our collaborators at University of Verona, were injected alone or with alginate, a bioactive extracellular matrix, provided by our partners at Italian Institute of Technology (IIT) in Genova, that should support the cells. The volume of alginate injected was chosen in order to optimize both the coverage of the injured area and the adequate support to the NSCs injected. The immunostaining analysis on microglia and astrocytes confirms the anti-inflammatory activity of alginate even in a very hostile context such as the injured epileptic brain. Our immunofluorescence data show that 9 weeks after transplantation cells are still alive and show an ongoing neuronal maturation process. These results offer important premises to achieve the goal of the project, supporting the possibility of reconstructing a functional neuronal circuit and implementing regenerative medicine with microelectronics and artificial intelligence.È consentito all'utente scaricare e condividere i documenti disponibili a testo pieno in UNITESI UNIPV nel rispetto della licenza Creative Commons del tipo CC BY NC ND.
Per maggiori informazioni e per verifiche sull'eventuale disponibilità del file scrivere a: unitesi@unipv.it.
https://hdl.handle.net/20.500.14239/13963