Promoting neuronal viability by mechano-regulation of neuron-to-neuron contacts

• Main Author: Li, Zhuyun
• Other Authors: Chen, Xiaodong
• Format: Thesis-Doctor of Philosophy
• Language: English
• Published: Nanyang Technological University 2018
• Subjects: DRNTU::Engineering::Materials::Biomaterials; DRNTU::Science::Biological sciences
• Online Access: http://hdl.handle.net/10356/73429
• Institution: Nanyang Technological University

Neurons are the longest-living terminally differentiated cells in the body. They can survive for an entire duration of human life while continually maintaining our reflexes, behavioral patterns, emotions and memories. Progressive depletion of long-lived nerve cells due to an imbalance between programmed cell death and relatively inefficient adult neurogenesis plays a central part in etiology of neurodegenerative diseases, stroke, and aging-related cognitive and motor disorders. Prolonging neuronal survival and developing artificial neural circuitries may help to develop novel therapies against neurondegenerative disorders, futuristic devices facilitating neural system regeneration, develop smart human prosthesis. The number of cells in developing neuronal networks in vivo is controlled by a balance between proliferation of progenitor cells and programmed death (apoptosis) of neurons. Neuronal death also contributes to network formation in a popular in vitro model where primary neurons from rodent brain are cultured on a 2D surface in a random manner. However, the underlying mechanism of this apoptosis process is not fully understood yet. This thesis aimed at finding how extracellular matrix affects neuronal cell behaviors and finding material fabrication ways to improve neuronal viability at early developmental stage. Cellular signaling through diffusible factors and direct cell-to-cell contacts are known to play a critical role in controlling cell viability. We, therefore, hypothesized that neuron survival in vitro could be improved using micro-patterned surfaces that impose stereotypic interneuron spacing and connectivity. In natural neuron develop systems, neurons extend their neurite unconstrained, with some guide thought growth factors diffused by other neurons and targeted cells. However, the guiding process has many disturbing factors and have large randomness. By providing a physical guide for neurite outgrowth, we build a road to direct neurite to reach their target. We proposed that with some design of the road network, neurons made contact with other neurons faster than on undersigned substrate. The accelerated connection process might increase localize survival factor concentration and cell-to-cell contact, and might active cell survival pathways. We designed a library of patterns where evenly spaced nodes are linked by different number of edges. We then fabricated this library using photolithography and micro-contact printing depositing cell-adhesive material in a predefined manner. When primary neuronal cultures were established on these patterned templates, the nodes tended to anchor neuronal somata whereas the edges functioned as tracks directing neurite outgrowth and promoting interneuronal connectivity. Notably, patterns with a large number of edges significantly improved neuronal survival as compared to random cultures. On the contrary, survival of neurons on patterns with few or no edges was significantly reduced compared to the random control. We analyzed the image to quantify cell-to-cell contact on different patterns and correlated them with cell survival data. We found that neuron cell survival positively correlates with connectivity. By using gene analysis methods, we found that several IEG (immediate early gene) are regulated on the patterned surface. And particular four genes are regulated by neuron-to-neuron contact. Among them, Nr4a1 genes can regulate neuronal survival. In conclusion, we did a systematic research on how designed surface topography can affect neuronal viability and find out the molecular biology mechanisms behind. We find early neuron-to-neuron contact can improve neuronal viability. Our research can improve current neuronal culture viability and give the field some hints on neuronal early apoptosis mechanisms.