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Restoration of motor dysfunction in vivo through nanomaterials based devices

Periodic Reporting for period 1 - NanoZfish (Restoration of motor dysfunction in vivo through nanomaterials based devices)

Reporting period: 2017-06-01 to 2019-05-31

Motor function is critical to human survival as it governs fundamental behaviours such as locomotion, breathing, speech, feeding and social interaction. Thus, even minor impairment of motor activity can severely hamper quality of life while major impairments, as occur in the neurodegenerative disorder amyotrophic lateral sclerosis (ALS), can be life-threatening. These conditions are characterized by lesions to the spinal cord, a region dedicated to the generation of motor behaviours such as walking and swimming. The spinal cord is composed of central pattern generating circuits, a series of evolutionarily conserved neural networks composed of rhythm-generating interneurons that transmit information directly to the motor neurons that execute muscle contractions. Disruption in the function of interneurons or motor neurons within these circuits can have severe consequences to motor performance and thus survival.
Over the past two decades, due to their unique combination of chemical and physical properties, carbon based nanomaterials have garnered increasing interest as tools for biomedical applications in the field of neuroscience. Among these, graphene possess the simplest structure, being formed by a mono-layer of carbon atoms. Other carbon based nanomaterials derive from it, such as graphene oxide (GO), a functionalized form of graphene containing carbon, oxygen, and hydrogen in variable ratios, and carbon nanotubes, cylindrically-shaped nanostructures composed of graphene sheets that are rolled to form hollow tubes. Recent evidence has showed that these nanomaterials are able to interact with cells of the nervous system and to modulate their function. This holds a great potential for the development of a novel class of nanomaterials based therapeutics for the rescue of defects in the brain. However, the effect of these materials on neuronal network in vivo and on animal behaviour has been poorly understood yet.
To investigate this issue, we have used larval zebrafish as an in vivo model. Early stage zebrafish are ideally suited for this purpose as they are amenable to a broad range of in vivo methodologies, including patch clamp electrophysiology to characterize the electrical activity of neurons, confocal imaging to examine neuronal morphology and behavioural analysis to study their locomotor activity. The purpose of this project was to use zebrafish models to determine whether carbon based nanomaterials could be exploited for the modulation of spinal neuronal activity and of correlated locomotor behaviour in vivo, with the final aim to explore the potential of these materials as novel therapeutics for the cure of nervous system pathologies.
Three types of carbon based nanomaterials were considered in this study: oxygen free graphene flakes (GF), small graphene oxide nanosheets (s-GO) and amino functionalised carbon nanotubes (f-CNT). A consistent part of the action was devoted to probe the in vivo biocompatibility of these nanomaterials. We observed that the anatomical development of larvae and the survival of neurons were not affected by injections of s-GO and GF in the spinal cord of animals.
However, the main outcome of this research is not merely a proof of biocompatibility. In fact, we observed that the intra-spinal delivery of s-GO was able to modulate selectively the excitatory transmission between neurons without affecting their survival and health. In vivo patch clamp recordings from spinal motor neurons performed two days after the treatment resulted in a strong and specific reduction in release of the main excitatory neurotransmitter of the brain, glutamate. This phenomenon was also detected in terms of a reduction in the excitatory signalling to motor neurons during fictive swimming (that is electrical activity that correlates with bouts of locomotor behaviour in paralysed fish). According to these findings, zebrafish intra-spinally injected with s-GO showed a decrease in their locomotor performance in behavioural experiments. Notably, the observed effects were specific for s-GO, since the treatment with GF did not affect synaptic transmission or locomotor activity of injected larvae.
Our results provide the first in vivo demonstration that s-GO, when injected intra-spinally, modulate synaptic signalling and this results in modifications of animal behaviour.
Thus, the main beneficiaries of these findings will be scientists and clinicians studying neurophysiology, neuropathology, neuropharmacology and biomedicine. However, due to the interdisciplinarity of this project, also people working in the field of materials science and engineering will be able to take advantage of such discoveries. They will be guided in the chemo-physical modification of nanomaterials to adapt them to the needs of life science scientists to develop together innovative devices for the cure of diseased brain.
The findings of this project were disseminated mainly through presentations to academic community. The largest public was reached at the FENS Forum of Neuroscience (Berlin, July 8-11th 2018; 7400 participants) thanks to a poster illustrating the results of the project. However, periodic presentations were given to smaller audience at the University of Leicester, where the research took place, and recurrent meetings were entertained with the partners of the project (University of Manchester), favoring the formation of a scientific network between neuroscientists and scientists of materials. A not specialized public of university and school students was targeted by dissemination of results during visits at laboratories.
NanoZfish has validated zebrafish as a model to study in vivo the effect of nanomaterials, and more generally of new therapeutic tools, with diverse degrees of resolution: on animal behaviour, on neuronal network activity and in terms of morphological interaction with neurons. Thus, this work represents a refinement of existing in vivo mammalian models and might impact on the 3Rs: Replacement, Reduction and Refinement programme, aimed to a more ethical use of animals for scientific purposes.
Significant progress has been made in characterizing a type of graphene derived nanomaterial, s-GO, as an innovative therapeutic strategy able to modulate excitatory synaptic transmission. For the first time, the effect of this nanomaterial on neuronal functionality was reproduced in vivo and the correlated outcome in terms of modification of animal behavior was characterized. This piece of information will serve for both research and clinical usage, potentially paving the way towards the applications of s-GO for the cure of nervous system pathologies in which excitatory signaling is aberrantly increased. Since many of these, such as Huntington’s, Alzheimer’s disease, epilepsy, pain and anxiety related disorders, have currently limited effective therapies, the use of s-GO might reduce the present high social and economic costs due to these diseases.