Abstract
Purpose :
Replacing retinal ganglion cells (RGC) in a degenerated optic nerve is not yet possible but may become feasible in the future through stem cell transplantation. Microfluidic fluid streams, presumably present in the optic nerve, are among the potential cues influencing speed and direction of axon outgrowth to its target. The largely unknown effect of microfluidics on RGC neurite outgrowth was studied.
Methods :
Primary cell cultures of postnatal rat RGC and cortical neurons were used. Cells were grown for 48h inside flow chambers and exposed for 24h to different flow rates (FR) inducing shear stress from 10-7 to 10-3 dyn/cm2. For inhibition of mechanosensitive calcium channels (MCC), I-RTX (TRPV1), HC-067047 (TRPV4), or GsMTx4 (Piezo1&2) was added. BAPTA-AM was used as intracellular calcium chelator. Cells density (CD), length of the longest neurite (LLN), length of all but the longest neurite (ALN), number of neurites (NN), and growth direction were assessed. The flow-to-non-flow ratio of each parameter was calculated and statistically tested with reference to the value 1 (no change).
Results :
Lower FR (10-6 dyn/cm2) significantly increased the LLN (ratio 1.3±0.16, p=0.0003), while inhibiting ALN (p=0.31). The higher FR (10-3 dyn/cm2) caused significant degeneration with reduced LLN and CD (ratio 0.6±0.06, p=0.0008; ratio 0.5±0.18, p=0.0035). However, no change was observed in cortical neurons at either FR (10-6 dyn/cm2: LLN, p=0.6; CD, p=0.3; 10-3 dyn/cm2: LLN, p=0.1; CD, p=0.07). Assuming that the effects were mediated by MCC the above-named inhibitors were tested at low and high FR. The LLN growth-promoting effect of low FR was inhibited by all MCC inhibitors (ratio not significantly different to 1 for Piezo1&2 and TRPV4; TRPV1 significantly shorter than control (1.1±0.05, control 1.3±0.16, p=0.02)). ALN even showed a trend towards longer neurites (ratio significantly higher than 1 for TRPV4 (ratio 1.2±0.14, p=0.046); not different for Piezo1&2 (p=0.2) and TRPV1 (p=0.3)). The degenerative effect of the higher FR on LLN and CD was returned to values not different to non-flow control by TRPV1&4 inhibition or BAPTA but not by Piezo1&2 inhibition.
Conclusions :
As opposed to cortical neurons, RGC are highly sensitive to extracellular microfluidic flow. Lower FR promote outgrowth of the presumed future axon and inhibit elongation of the future dendrites, while higher FR induce neurodegeneration. This is mediated by MCC.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.