Influence of cyclic mechanical strain on tissues of the central nervous system

Abstract

Although the brain is the softest tissue of the human body, cells embedded in the brain are responding to physiological mechanical cues. Tissue homeostasis is, therefore, not only dependent on chemical cues (e.g. growth factors), but also integrates the mechanical input that brain cells can sense from their physical microenvironment. The softness of the brain tissue, as well as the protective role of thick meninges and the hard skeletal skull, are the main reasons why mechanobiology for brain cells remained relatively unattended. Brain cells, in general, were regarded as mechanosensitive elements rather than seeing mechanical cues as an essential part of brain physiology. Recent studies, however, show an imposing involvement of physical cues, such as stiffness alterations and topographical cues. <br /> As brain cells are also subjected to cyclic deformation due to the highly vascularized character of the brain tissue, cyclic mechanical strain is the focus of this thesis. Cyclic mechanical strain might play a relevant role in brain physiology as mechanical strain has shown to influence relevant biological signaling processes and gene expression in other cell types. Further, each cell type found in the brain has its own unique cytoskeletal arrangement. Cytoskeletal systems are involved in mechanotransduction and are the first recipients when cells are exposed to mechanical strain. A different cytoskeletal arrangement in each cell type is further highlighting the question of how mechanoresponses may differ between each individual cell type. <br /> In the context of this dissertation, cells found in the brain were subjected to uniaxial cyclic strain within different developing stages. Therefore, neural stem cells, premature neuronal cells and astrocytes, as well as developed neuronal networks, and astrocytes were exposed to cyclic strain and mechanoresponses were analyzed. All cell types show striking differences in how they handled mechanical forces and revealed individual patterns of cytoskeletal alterations. Two different mechanical stimuli were used to analyze individual responses of the cells. The first part of this work was focused on immediate cell response to the first cycles of substrate deformation. In the second part, all cell types were analyzed according to their long-term adaptation to cyclic mechanical strain. <br /> As an immediate response, neural stem cells revealed a reduced migration velocity and directional extensions of cell processes along the axis of uniaxial strain. Quantitative orientation analysis confirms the parallel alignment of neural stem cell extensions even after long-term stretch experiments. The parallel direction of cell alignment was sustained for neural stem cells that have been committed to an astrocyte phenotype when stretched during the differentiation process, while a neuronal commitment revealed a more random distribution with a slight shift towards a perpendicular direction. When NSCs were subjected to cyclic strain during differentiation, lineage commitment was not altered. However, neural stem cells were more quiescent when subjected to cyclic strain as less proliferative cells were found on stretched chambers. <br /> The neuronal immediate response analysis in live-cell stretch experiments, revealed a drastic response of neuronal cells as they retract their branches within the first cycles of stretch. Such retraction was explicit when neuronal cells have been developed to neuronal networks on the elastomer. With more elongated neuronal branches, such retraction was accompanied by the formation of retraction bulbs filled with destabilized cytoskeletal proteins. A prolonged cyclic stretch triggered an adaptation process and allowed the neuronal cell to regrow their branches even under cyclic mechanical strain. Moreover, live-dead analysis after long-term stretch revealed that neuronal cells can survive long-term mechanical loads and did not show any altered cell vitality. Long-term stretch revealed a clear mechanoresponse and growth of neuronal branches in perpendicular direction. In addition, stretched neuronal cells showed an induced outgrowth with a higher number of branches, an increased sum length, and an enlarged growth cone. <br /> Mature astrocytes isolated from postnatal rat pups did not show any directional mechanoresponse. They thereby behaved contrary to the cells that were differentiating from neural stem cells and committed towards an astrocyte phenotype. In a co-culture of astrocytes and neuronal cells, astrocytes revealed a mechanoprotective role and neuronal cells that grew on top of these astrocytes did show less strain-induced responses compared to cyclically stretched single neuronal cell cultures.