The loss of connection between muscle and nerve is a crucial biological mechanism severely impaired in many pathologic conditions as aging and neuromuscular disorders. In our laboratory we recently developed an innervated in vitro engineered muscle tissue, the X MET, obtained from murine skeletal muscle primary culture, which closely mimics the architecture and the function of skeletal muscle. Starting from the techniques already used in literature on in vitro muscle tissues, the aim of this project is to develop a measurement system and a testing protocol for the functional characterization of the X-MET's neuromuscular junction. The technique is based on the comparison between muscle contractile response to direct membrane electrical stimulation and to nerve electrical stimulation. During the experiments the tissue will be mounted between the lever arm of a length actuator and a micro-force transducer. Two platinum electrodes connected to a pulse stimulator will be located parallel to the tissue to elicit tissue contraction through direct stimulation. For nerve stimulation, the nerve will be sucked in a suction electrode and pulses will be delivered through the nerve using a second pulse stimulator. Moreover, an ad-hoc stimulation protocol will be developed. Results of this research will provide important information to improve techniques and protocols for the realization of the innervated X-MET. Once obtained the characterization of the NMJ, the innervated X MET can be used as three dimensional model of an in vitro muscle tissue, thus simplifying the study of complex cellular processes. Moreover, innervated X METs should be realized starting form cultures of pathological mice, thus offering the possibility to elucidate fundamental issues related to the impaired communication between muscle and nerve.
In this project, a measurement system for the functional characterization of the neuromuscular junction of an innervated ex vivo muscle engineered tissue (X-MET) is proposed.
Results of this research will be a useful tool to provide important information to improve techniques and protocols for the realization of the innervated X-MET, in order to obtain a three dimensional construct which even more closely mimics the physiological structure of the motor unit.
Once obtained the characterization of the NMJ, the innervated X MET can be used as three dimensional model of an in vitro muscle tissue, thus simplifying the study of complex cellular processes. In fact, three dimensional cultures more closely represent physiological conditions with respect to two dimensional constructs, and they more faithfully reproduce the interaction between adjacent cells. Moreover, three-dimensional cultures can be considered a viable alternative to the use of animal models, thus reducing their use in biomedical research and simplifying the analysis of complex system, such as the study of neuromuscular junction here proposed.
One important aspect of the innervated X-MET is the possibility to realize pathological NMJ model starting from primary cultures and phrenic nerve of pathological animal models, as for example the SOD1G93A mice, a model of Amyotrophic Lateral Sclerosis, in order to better understand the mechanisms which lead to the impairment of the neuromuscular junction this fatal disease. In fact, studies on ALS animal models showed that the physiological connection between muscle and nerve is severely impaired [1], [2], suggesting that the NMJ might be a direct target of the SOD1 mediated toxicity. However, although different pathogenic mechanisms have been proposed to account for ALS [3], several questions are still open and ALS remains a fatal disease.
Other studies on SOD1G93A mice reported a deficit of the extensor digitorum longus (EDL) in generating maximum force after the onset of the pathology [4], [5] and a trend toward lower values of maximum specific force in diaphragm strips [5], when compared to wild type littermates. However those studies did not determined whether the alterations were due to changes either in muscle or in neuromuscular transmission. This is an important aspect which could be investigated using an innervated X-MEX realized through hybrid cultures. In fact, the engineered muscle tissue could be realized using primary cultures of SOD1G93A mice and phrenic nerves of wild type mice and vice-versa.
Therefore the proposed study will have a direct impact on the field of research of neurodegenerative diseases, and will contribute to elucidate fundamental issues related to the impaired communication between muscle and nerve.
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[2] Musarò, A., "Understanding ALS: new therapeutic approaches.," FEBS J., 2012.
[3] Rosen, D. R., T. Siddique, D. Patterson, D. A. Figlewicz, P. Sapp, A. Hentati, D. Donaldson, J. Goto, J. P. O'Regan, and H. X. Deng,"Mutations in Cu/Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis.," Nature, 1993.
[4] Derave, W., B. L. Van Den, G. Lemmens, B. O. Eijnde, W. Robberecht, and P. Hespel, "Skeletal muscle properties in a transgenic mouse model for amyotrophic lateral sclerosis: effects of creatine treatment.," Neurobiol. Dis., 2003.
[5] Hwee, D. T., A. Kennedy, J. Ryans, A. J. Russell, Z. Jia, A. C. Hinken, D. J. Morgans, F. I. Malik, and J. R. Jasper, "Fast skeletal muscle troponin activator tirasemtiv increases muscle function and performance in the B6SJL-SOD1G93A ALS mouse model.," PLoS One, 2014.