Abstract
Background: Low-intensity pulsed ultrasound (LIPUS) has been used in clinical studies. But little is known about its effects on the central nervous system (CNS), or its mechanism of action. Retinal ganglion cells (RGCs) are CNS neuronal cells that can be utilized as a classic model system to evaluate outcomes of LIPUS protection from external trauma-induced retinal injury. In this study, we aim to: (1) determine the pulse energy and the capability of LIPUS in RGC viability, (2) ascertain the protective role of LIPUS in optic nerve (ON) crush-induced retinal injury, and 3) explore the cellular mechanisms of RGC apoptosis prevention by LIPUS.Methods: An ON crush model was set up to induce RGC death. LIPUS was used to treat mice eyes daily, and the retina samples were dissected for immunostaining and Western blot. The expression of yes-associated protein (YAP) and apoptosis-related proteins was detected by immunostaining and Western blot in vitro and in vivo. Apoptosis of RGCs was evaluated by TUNEL staining, the survival of RGCs and retained axons were labeled by Fluoro-gold and Tuj1 antibody, respectively. Rotenone was used to set up an in vitro cellular degenerative model and siYAP was used to interfering the expression of YAP to detect the LIPUS protective function.Results: LIPUS protected RGC from loss and apoptosis in vivo and in vitro. The ratio of cleaved/pro-caspase3 also decreased significantly under LIPUS treatment. As a cellular mechanical sensor, YAP expression increased and YAP translocated to nucleus in LIPUS stimulation group, however, phospho-YAP was found to be decreased. When YAP was inhibited, the LIPUS could not protect RGC from caspase3-dependent apoptosis.Conclusion: LIPUS prevented RGCs from apoptosis in an ON crush model and in vitro cellular degenerative model, which indicates a potential treatment for further traumatic ON injury. The mechanism of protection is dependent on YAP activation and correlated with caspase-3 signaling.
Highlights
Traumatic injury-induced optic nerve (ON) degeneration and glaucoma-triggered possessive retinal ganglion cell (RGC) death are the main challenges of vision loss
The energy of Grade 1 ranged from 40.5 ± 1.857 to 40.3 ± 0.919 mW/cm2 from 2nd to 10th-min duration of measurement, Grade 2 ranged from 123 ± 2.781 to 110.667 ± 3.138 mW/cm2, Grade 3 ranged from 230.333 ± 2.985 to 191 ± 1.915 mW/cm2, and Grade 5 ranged from 427.167 ± 8.345 to 354.647 ± 10.467 mW/cm2
These results are in consistent with the caspase-3 results (Figure 7F). It implies that Yes-associated protein (YAP) is the main cellular mechanosensitive factor in protecting RGCs from apoptosis (Figure 7G, ∗∗P < 0.01, n = 6). These findings indicated that YAP is essential for low-intensity pulsed ultrasound (LIPUS) stimulation and pivotal for LIPUS-induced RGC survival by regulating procaspase-3/cleaved caspase-3 signaling
Summary
Traumatic injury-induced optic nerve (ON) degeneration and glaucoma-triggered possessive retinal ganglion cell (RGC) death are the main challenges of vision loss. In peripheral nervous system (PNS), noninvasive low-intensity ultrasound was used to sustain neuronal soma survive and to promote peripheral nerve regeneration with functional recovery by stimulating brain-derived nerve growth factor (BDNF) release (Ni et al, 2017). In vitro study demonstrated that nerve growth factor (NGF) combined with low-intensity pulsed ultrasound (LIPUS) stimulation may have some effects on PC12 cell neurite outgrowth (Zhao et al, 2016). We aim to: (1) determine the pulse energy and the capability of LIPUS in RGC viability, (2) ascertain the protective role of LIPUS in optic nerve (ON) crush-induced retinal injury, and 3) explore the cellular mechanisms of RGC apoptosis prevention by LIPUS
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