Poster Z16 - Fluid shear stress promotes glial-mediated neurotoxicity

Introduction: The convective flow of interstitial fluid within tissue is a ubiquitous physiological process. This flow brings nutrients into tissue, removes cellular waste, and exerts shear stresses on cells to instruct cell function. Many neurological and neurodegenerative disorders are associated with disruption of interstitial fluid flow pathways and patterns within neural tissue, but the role fluid shear stress plays in mediating or propagating neurological disease is currently not well understood. Better characterizing how cells of the central nervous system sense and respond to heightened fluid shear stress will enhance our understanding of neuropathology and may reveal new therapeutic targets. Here, we investigate how astrocytes and microglia respond to heightened fluid shear stress and the downstream effects on neurons.

Materials and

Methods: Immortalized human microglia (ABM) and human cortical astrocytes (ScienCell) were maintained in Dulbecco’s Modified Eagle Medium with 10% fetal bovine serum (FBS) and Astrocyte Medium (AM) supplemented with FBS and astrocyte growth supplement, respectively. For experimentation, cells were plated at 1x10^5 cells per well in 6-well collagen-coated tissue culture-treated plates. After 24 hours, cells were serum starved in AM supplemented with B-27 and N-2 Supplement. Experimental cells were exposed to laminar shear stress on an orbital shaker at 100 rpm, equivalent to 0.1 dynes/cm^2, or low pathological shear stress. Control cells remained static. After 18 hours, media was collected and stored at -20℃. To test the role of lipid components, glial cell conditioned media samples were filtered with activated charcoal for two hours on a tube rotator, then passed through a 0.22 μm filter. To test the role of protein components, conditioned media were boiled at 97°C for 5 minutes then added to neuronal-like cells. Neuronal-like cells were generated by differentiating 1.15x10^4 SH-SY5Y neuroblastoma cells (ATCC) per 48-well in Neurobasal medium supplemented with GlutaMAX, B-27 and N-2 supplements, and 10 μM retinoic acid for 5 days. After sufficient neurite outgrowth was visible, 200 μL of glial cell conditioned media was added to each well. Cells were incubated with conditioned media for three days, then fixed with 4% paraformaldehyde. Neurite outgrowth was evaluated via immunocytochemistry with mouse anti-beta III tubulin (Abcam) and quantified using the NeuronJ plugin on ImageJ. Statistical comparisons were performed in GraphPad Prism 10.2.2.

Results, Conclusions, and Discussions: Shear-conditioned media from both microglia and astrocytes significantly reduced the average neurite length compared to static-conditioned media controls (Fig. 1A). In order to investigate the nature of the molecule released by glia responsible for the neurotoxicity effect observed, we boiled the glial-conditioned media to denature any proteins therein. This process did not eliminate the neurotoxic effect for astrocyte- or microglia-conditioned media (Fig. 1B), suggesting the neurotoxic factor is not proteinaceous. We alternatively filtered the glial-conditioned media with activated charcoal to strip any lipophilic molecules present. This process eliminated the reduction in neurite length in shear-conditioned media compared to static for media from both astrocytes and microglia (Fig. 1C-D), indicating the presence of a neurotoxic lipid molecule(s). We also found that blocking the sphingosine-1-phosphate receptor 3 in neurons preserved neurite length (not shown), and we are currently working to quantify sphingosine levels in the conditioned media. Additionally, we have found increased expression of some, but not all, classical markers of activation in the shear-conditioned astrocytes and microglia (not shown).
Exerting low pathological levels of shear stress on microglia and astrocytes mediates a neurotoxic effect on neuronal-like cells, which is alleviated by charcoal stripping of the lipophilic molecules from the conditioned media. We therefore hypothesize that the molecule or pathway responsible for the neurotoxic effect is lipophilic. Blocking experiments suggest this response, at least in part, may be mediated by the sphingosine-1 phosphate (S1P) - sphingosine-1 phosphate receptor 3 (S1PR3) axis, which was previously shown to promote neurite retraction. Given the acknowledged role of this axis in neurodegenerative disorders, our results suggest that shear-induced activation of the S1P-S1PR3 axis may play a role in mediating or propagating neuropathology.

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