Rat embryonic hippocampus and induced pluripotent stem cell derived cultured neurons recover from laser-induced subaxotomy

Abstract

Abstract. Axonal injury and stress have long been thought to play a pathogenic role in a variety of neurodegenerative diseases. However, a model for studying single-cell axonal injury in mammalian cells and the processes of repair has not been established. The purpose of this study was to examine the response of neuronal growth cones to laser-induced axonal damage in cultures of embryonic rat hippocampal neurons and induced pluripotent stem cell (iPSC) derived human neurons. A 532-nm pulsed Nd∶YVO 4 picosecond laser was focused to a diffraction limited spot at a precise location on an axon using a laser energy/power that did not rupture the cell membrane (subaxotomy). Subsequent time series images were taken to follow axonal recovery and growth cone dynamics. After laser subaxotomy, axons thinned at the damage site and initiated a dynamic cytoskeletal remodeling process to restore axonal thickness. The growth cone was observed to play a role in the repair process in both hippocampal and iPSC-derived neurons. Immunofluorescence staining confirmed structural tubulin damage and revealed initial phases of actin-based cytoskeletal remodeling at the damage site. The results of this study indicate that there is a repeatable and cross-species repair response of axons and growth cones after laser-induced damage.

Defective Transcytosis of APP and Lipoproteins in Human iPSC-Derived Neurons with Familial Alzheimer’s Disease Mutations

“…To ensure that we were quantifying staining in axons and not dendritic processes, we made use of microfluidic compartments, which separate axons from the bulk somatodendritic population (Figure 1E) (Niederst et al, 2015; Selfridge et al, 2015; Taylor et al, 2006). Neurons grown in microfluidic devices extend long processes into the axonal space that do not stain for the somatodendritic marker Map2 and that do stain for the axonal marker neurofilament-H (SMI31) (Selfride et al, 2015) (Figure 1E and Figure S1A). We observed that PS1 ΔE9 axons grown in microfluidic devices have decreased axonal APP puncta and APP puncta intensity (Figure 1D); this decrease is sensitive to PS1 ΔE9 gene dose.…”

A model for traumatic brain injury using laser induced shockwaves

“…After imaging, the time series were qualitatively sorted into (1) positive responses, where axons showed significant repair response following damage, (2) neutral responses, where the growth cones recovered without significant changes in apparent dynamics, and (3) negative responses, where neurons did not recover from damage. These time series were then quantitatively assessed using the wavelet decomposition algorithm, where a region of interest, generally the growth cone, was selected and the wavelet coefficients for each frequency band measured 7 .…”

“…All cells with less than 75 percent decrease in activity were independently categorized as having neutral or positive responses. Based on this correlation, the dynamics of the wavelet coefficients were judged to be a meaningful indicator of the overall health and activity of the growth cone 7 . The algorithm was later applied to the measurement and classification of growth cone responses to shock wave damage.…”

“…This finding is consistent with the observation that growth cones tend to shuttle material towards damage sites along their own axon, due to the abundance of actin rich structures throughout the growth cone. Additionally, tubulin appears to vacate damage sites along the axon for a sustained period of time before recovering along the axon as the recovery process finishes 7 .…”

“…One major obstacle to this goal is the difficulty of maintaining healthy, active growth cones, especially following LifeAct RFP infection. Through out sub-axotomyand shock wave experiments, neurons were seen to be most active at three days following plating 7 . After infection, however, approximately three days were necessary for the cells to express sufficient protein for imaging.…”