Mechanical strain influence on functional signaling of neurons in vitro

Abstract

Neuronal cells are embedded in the soft brain tissue and are protected by the thick meninges, as well as the rigid skull bones. Despite being highly sensitive and vastly protected, neurons are prone to internal and external mechanical forces. Many internal tensile loading scenarios affect neurons during development, growth and blood flow. Moreover, neurons are exposed to be strained externally by regular body movements or to be severely injured as a result of serious falls, accidents or tumor formation. In specific cases neuronal tissue could be dissected where intercellular connections get an entire cut and may cause cell death for some neurons in a network, such as the case of brain surgery to remove a tumor or to implant a neurostimulator device. However, limited experimental data is available for the mechanobiology of brain cells and it remained quite unattended. <br /> Beside the chemical and molecular cues, some studies reported the importance of the mechanical input in nervous tissue homeostasis. Latest studies, show the key role of the physical microenvironment, such as topographical cues and stiffness alterations. Moreover, reports on physiological cyclic mechanical strain show the potential to accelerate axonal growth. While strain induced responses on the molecular and structural levels has been identified, much less is known about the consequences of these changes to neuronal functionality. As neurons are the basic functional units in transmitting information in the nervous system, the functional behavior of neurons under mechanical strain is the focus of this dissertation. In the context of this thesis, primary cortical neurons were functionally characterized upon exposure to physiological and traumatic uniaxial stretch conditions within different developing stages. Also, spontaneous signaling events were characterized upon single cellular compartment loss induced by laser ablation. The dynamic changes in cells was determined by recording spatial and temporal changes in Ca²⁺ concentrations ([Ca²⁺]) of single-cells and on synchronized network level using the Cal-590 AM Ca²⁺ indicator. The focus here is on both the immediate cell response and to their long-term adaptation to mechanical strain. This work shows that neurons are robust and can functionally tolerate cyclic stretching of up to 30% strain by keeping active communication between cells no matter if strain is applied at different periods during neuronal network formation. Simulating traumatic brain injuries using rapid stretch pulses identified a threshold for functional impairment of about 60% with a high ability to adapt and restore cellular connectivity with time. In spite of the broad range of assessed strains, cells keep maintained without further effects on cell viability, inflammatory responses or synapse formation. Co-cultures with astrocytes revealed more stable and better communicating networks while functional responses to strain remained unaffected. <br /> Laser-induced death of single cellular compartment resulted in an increased intracellular calcium concentration [Ca²⁺]i in neurons surrounding the ablated point. The [Ca²⁺]i-increase was distance-dependent as closely connected neurons to the ablated cell were highly affected by Ca²⁺ inflow compared with other cells. Furthermore, the increased [Ca²⁺]i caused a temporarily interruption in neuronal spiking activity that recovered gradually with time. In spite of the distance-dependent alterations, the overall network functionality was unaffected and the level of connectivity was maintained in the long-term analysis. <br /> Taken together, this research work illustrates the internal dynamic responses of neurons to multiple loading conditions and to single-cell death. These data will be advantageous in developing more effective neuronal tolerance criteria to injury. Understanding and pushing the limits of nerve stretch holds tremendous potential for tissue engineering efforts to prevent nervous system injury and facilitate nerve repair.