Abstract
Peripheral nerves are continuously subjected to mechanical strain during everyday movements, but excessive stretch can lead to damage and neuronal cell functionality can also be impaired. To better understand cellular processes triggered by stretch, it is necessary to develop in vitro experimental methods that allow multiple concurrent measurements and replicate in vivo mechanical conditions. Current commercially available cell stretching devices do not allow flexible experimental design, restricting the range of possible multi-physics measurements.Here, we describe and characterise a custom-built uniaxial substrate-straining device, with which neurons cultured on aligned patterned surfaces (50 µm wide grooves) can be strained up to 70% and simultaneously imaged with widefield and confocal imaging (up to 100x magnification). Furthermore, direct and indirect electrophysiological measurements by patch clamping and calcium imaging can be made during strain application. We characterise the strain applied to cells cultured in deformable wells by using finite element method simulations and experimental data, showing local surface strains of up to 60% with applied strains of up to 25%. We also show how patterned substrates do not alter the mechanical properties of the system compared to unpatterned surfaces whilst still inducing a homogeneous cell response to strain.The characterisation of this device will be useful for research into investigating the effect of whole-cell mechanical stretch on neurons at both single cell and network scales, with applications found in peripheral neuropathy modelling and in platforms for preventive and regenerative studies.
Highlights
The peripheral nervous system (PNS) is tollerant to continuous mechanical stresses during ordinary movements, but supraphysiological extension can lead to injury
Differentiated F11 cell populations were strained by 45% and whole-cell current and voltage clamp recordings were acquired while the stretch was maintained, to demonstrate the compatibility of our device with simultaneous direct electrophysiology measurements
Inwards and outwards currents and induced action potentials were recorded from deformed cells (Fig. 3), demonstrating how this system can be used to record neural electrophysiology during acute cell deformation to examine the mechanisms involved in immediate mechanotransduction and primary ion channel and membrane damage
Summary
The peripheral nervous system (PNS) is tollerant to continuous mechanical stresses during ordinary movements, but supraphysiological extension can lead to injury. For example, is caused by excessive stretching of infant heads and arms during labour inducing loss of sensation and abnormal motor function in 0.1% of births in the US [1]. Neuron deformation has been shown to alter ion channel functionality [2], depress neural network activity [3], and cause cytoskeletal damage and collapse [4], implicating mechanotrans-. To better understand the mechanisms involved, cell-scale in vitro models reproducing the mechanical environment of the PNS are required. Ex vivo animal tissue does not allow for the investigation of neural cell response in isolation, so in vitro experimental devices have been designed to investigate a wide variety of biological responses to mechanical stimuli, but none for PNS modelling (see review by Kamble et al [5] )
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