Abstract
Purpose :
Retinal ganglion cells (RGC) are mechanosensitive, which is considered a feature driving degeneration. We have recently shown that 1) cultured RGC are far more sensitive to fluid flow induced shear stress than other neuronal cultures and 2) that very low (microfluidic) flow rates over 24h can promote neurite outgrowth. Through live imaging of RGC and longitudinal observation we now investigated the temporal dynamics of flow-induced changes.
Methods :
Primary cultures of RGC from postnatal rats were incubated in channel slides and exposed to either microfluidic flow (10-6 dyn/cm2 shear force) or a higher flow rate (10-3 dyn/cm2). Individual cells were followed for 8 h using live cell imaging. Length of the longest neurite (LLN) was measured. Mitochondria were stained with MitoTracker Red CMXRos and mitochondrial fluorescence in the longest neurite (MLN) was determined. The mechanosensitive calcium channels TRPV1 and 4 were pharmacologically inhibited.
Results :
High shear stress led to a significant reduction of mitochondrial fluorescence within 1h (MLN relative to baseline 0.92±0.09, p=0.003, n=14), followed by retraction of the longest neurite from 3h on (p=0.016). After 8h 64% neurites had retracted (median relative LLN 0.47 (0.01, 0.99 interquart. range (IQR)), p=0.005). Under TRPV1&4-channel inhibition the relative LLN of the neurites was not significantly different from baseline (rel. median 0.79 (0.23,1.36; n=18; p=0.17), less neurites retracted (50%) and more enlongated (39% vs 14% w/o inhibitors)). However, MLN reduction was not attenuated (p=0.18).
Microfluidic shear force showed a trend towards an increase of median rel. LLN compared to no-flow (0.95 vs 0.85, p=0.68), while the amplitude of retraction/elongation increased (IQR 0.5 vs 0.31). The MLN remained unchanged. TRPV-inhibition reduced the amplitude of retraction/elongation (IQR 0.5 vs 0.19). Retraction was attenuated significantly (median rel LLN 0.91 vs. 0.98, p=0.02) and elongation showed a trend towards attenuation (1.26 vs. 1.17, p=0.44).
Conclusions :
The neurite-degenerative effect of high shear stress could be well observed and possibly turned into a growth promoting effect in some RGC by TRPV inhibition. Mitochondria were early affected. Very low shear stress, however, showed a more disparate effect with slow kinetics. TRPV inhibition seemed to attenuate both, longest neurite elongation and retraction.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.