Devising techniques to probe the mechanical response of axons.

Abstract

Axons are long tubular structures of neurons that span in centimetres in brain and respond to physical stimuli through physiological changes. The ability of axons to withstand deformations and maintain their unusual shape is due to an elaborate cytoskeleton (complex network of interlinking protein filaments in cells) arrangement. A study in 2013 using stochastic optical reconstruction microscopy (STORM) identified the presence of nanoscale organisation of actin-spectrin rings in axons. The periodic arrangement includes filamentous actin (F-actin) rings separated by spectrin tetramers that can fold and unfold [1]. Cell stretching methods are used to probe the change in periodicity of the membrane-associated actin-spectrin cytoskeletal complex on a stretched axon. The objective of this project is to build a customised PDMS based stretching system meant for culturing and stretching, which is also expected to be amenable to be used in a super-resolution imaging system. The device is to be used to observe the membrane periodic skeleton under applied strain and to observe the effect of tension built up during compressive load at zero applied strain. Calculation and measurement of strain are important for both characterisation of the device as well as in the analysis of the cytoskeleton. Simulation through finite element modelling is carried out to estimate the amount of stress required to achieve optimum strain in a controllable fashion. From the predicted strain map, the distribution of strain on the PDMS sheet is estimated and design of device is optimised in steps of experiments. The optimised procedure for culturing neuron is also proposed in this study. The thickness of PDMS sheet and curing ratio were also optimised as 2-3mm thick and 1:9 ratios, respectively. The simulation and experimental trials found that curing ratio and friction in various parts of the device play a crucial role in maintaining uniform strain during stretching. Compressive loading process is introduced in the design of the device. It overcomes the issues such as maintaining constant strain and the possibility of relaxation of PDMS during the incubation time. The characterization of the device shows that the variation in strain along the surface is less than 1% is achieved. Super-resolution imaging using the devices is yet to be done.