Nanoscale Cue for Neural Cell Systems

a. Nanoscale Cues for Regenerative Neural Cell Systems

         The hypothesis that there is a fundamental set of nanoscale physical properties that serve as the primary cues for the re-establishment of cell systems that rely on extracellular matrices in their native environments, and that these may be as potent as better known biochemical and growth factor cues, is investigated in collaborative partnership with Rutgers University Bioengineering. A set of five potentially global cues: elasticity, surface polarity, and nanofibrillar matrix topography, in terms of three synergistically linked properties: surface roughness, nanofiber curvature, and mesh density, was identified and scanning probe recognition microscopy was used in concert with quantitative immunocytochemistry, super-resolution microscopy (newly available) and blot techniques to investigate cell responses to quantitatively described cue sets. Three additional culture surfaces were utilized as comparative cue sets and astrocytes treated with dBcAMP were used to simulate traumatic brain injury (TBI), while untreated astrocytes were used to approximate quiescence. A key concept was that cells respond to cue sets rather than individual properties. Cluster analysis was used to define nanophysical property sets and to assess degree of overlap. Key research findings to date include: (1) Cluster analysis demonstrated that the four cue set environments were non-overlapping and therefore presented quantifiably different trigger sets to cells. (2) Two nanophysical cue sets that triggered pathological responses were identified and quantitatively described for the first time. One of these induces the glial scar and TNT formation response pertinent to the proposed research. (3) The dBcAMP-treated astrocytes modulated by quantified nanofibrillar scaffold cues remained permissive across multiple protein markers: glial fibrillary acidic protein (GFAP), tubulin, actin, Rho GTPases: Cdc42, Rac1 and RhoA, and chondroitin sulfated proteoglycans (CSPGs).

b. Scanning Probe Recognition Microscopy

         SPRM is a new investigative capability, developed by Profs. Ayres and L. Udpa during NSF GOALI CMMI-0400298 and NSF SGER BES-0225805. In SPRM, a scanning probe microscope system is given the ability to auto-track on regions of interest through incorporation of recognition-based tip control. The recognition capability is realized using algorithms and techniques from computer vision, pattern recognition and signal processing fields. Adaptive learning and prediction are also implemented to make the detection and recognition procedure quicker and more reliable. It is applicable to any scanning probe microscopy investigation. An offline version for wider community access is under development in 2021.

         In the nanoscale cues investigations, SPRM uniquely allows auto-tracking and nano-indentation along individual nanofibers. An example of SPRM auto-tracking along the individual nanofibers within a user-defined bounding box (dashed) is shown in the figure. Arrows indicate the order and directions of the real-time motion along individual nanofibers that would be seen during SPRM live imaging.

         SPRM nanoscale property measurements along individual nanofibers are compiled into a statistical representation of the nanofibrillar matrix as a whole that provides ranges and distributions as well as mean and mode values for nanoscale properties. The difference between the ‘corrugation roughness’ experienced over a cell soma and the along-nanofiber surface roughness experienced by a receptor in a cell process is clear from the figure.

c. Tunneling Nanotubes

         Tunneling Nanotubes (TNTs) are a newly identified mechanism for the establishment of long-distance intercellular communication networks and are short-lived, highly efficient transporters for pathogens including viruses and calcium ions imbalances associated with viruses and immune reactions. Our work in nanoscale cues for regenerative neural systems identified a cue combination that provoked an apparent TNT response that strategically demonstrated the major variations in TNT structures and protein compositions reported to date. This work is under review in 2021 and we will post additional work and exciting images as soon as possible!