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Frank Kung, Abram Axelrod, Dionicio Siegel, Ellen Townes-Anderson; Rod Photoreceptor neuritic sprouting is responsive to Sema3A. Invest. Ophthalmol. Vis. Sci. 2013;54(15):4183.
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© ARVO (1962-2015); The Authors (2016-present)
Sema3A is known to increase in CNS injuries and disease including retinal injury. In addition, during retinal injury, rod photoreceptors form long neuritic sprouts towards the inner retina where Sema3A increases. Therefore, photoreceptors may be responsive to Sema3A. Our hypothesis is that Sema3A is attractive to injury-induced rod photoreceptor growth.
In this study, we utilized a microspritzer to determine if rod cells respond to gradients of Sema3A. Isolated rod photoreceptors with intact terminals were obtained from dissociated salamander retina. Rod cells were kept from 0-3 DIV. Gradients were produced by pulsing 25 µg/ml Sema3A-FC chimera from a micropipette with a tip inner diameter of ~ 1 µm. Pressure was set to 3-4 PSI, with an ejection time of 30 milliseconds at 1 Hz for 24 hours. The pipette was placed 100 µm away at a 45° angle from the axonal pole.
After application of gradients of Sema3A, the trajectory of the longest neurite occurred on average at an angle of 56° away from the gradient of Sema3A while a PBS gradient produced a trajectory at an angle of 171° away from the pipette (p<0.05 using the Watson's two sample U-squared test). Using Scholl analysis, the growth in neurite crossings after 24 hours decreased to 1.39 under Sema3A application from 5.73 under PBS application (p<0.005). This effect could be reversed using a heat-inactivated Sema3A (p<0.05). Finally, cell polarization, determined by the position of a line between the centers of the nucleus and ellipsoid, changed by ±23.0° under a Sema3A gradient while a PBS gradient only caused a ±10.1° fluctuation in polarization (p<0.05). This effect was reversed with the application of 5 µM Xanthofulvin, a Sema3A specific inhibitor as well as with heat-inactivated Sema3A.
Rod cells respond to gradients of Sema3A with multiple effects on growth. Compared to control, neuritic extension was preferentially toward the Sema gradient and cytoskeletal plasticity within the cell body (which modulates polarity) increased while overall growth decreased. Sema3A may therefore affect the neuritic sprouting of photoreceptors in injured or diseased retina.
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