Department of Ophthalmology, University Medical Center, Goettingen, Germany; Third Institute of Physics, Biophysics, Georg-August-University, Goettingen, Germany; Department of Ophthalmology, University Medical Center, Rostock, Germany.
Department of Ophthalmology, University Medical Center, Goettingen, Germany.
Exp Eye Res. 2024 Dec;249:110116. doi: 10.1016/j.exer.2024.110116. Epub 2024 Oct 5.
In vitro primary cell culture models of retinal ganglion cells (RGC) are widely used to study pathomechanisms of diseases such as glaucoma. The biomechanic interaction with the culture substrate is known to influence core cellular functions. RGC cultures, however, are usually grown on rigid plastic or glass substrates. We hypothesized that soft polyacrylamide gel substrates may alter survival and neurite outgrowth of primary cultured RGC. Primary retinal cultures from postnatal (day 1-6) Wistar rats were grown on glass coverslips or polyacrylamide (PA) gel substrate with different Young's elastic moduli (0.75, 10 or 30 kPa). Substrates were coated with Poly-l-lysine and/or laminin. RGC were immunostained with anti-beta-III-tubulin. Total neurite length, growth cone morphology, RGC density, mitochondrial morphology and transport as well as pro-survival pathways (Erk1/2, Akt, CREB) were assessed. PA gel substrates of E = 10 kPa significantly increased the total neurite length by factor 1.5 compared to glass (p = 0.02). The growth cone area was significantly larger by factor 5.3 on 30 kPa gels (p = 0.01). The presence of a substrate coating was more important for neurite outgrowth and RGC survival on PA gels (poly-l-lysine > laminin) than on glass. Neither mitochondrial morphology and motility nor the activation of pro-survival pathways significantly differed between the four substrates. PA gel substrates significantly enhanced RGC neurite outgrowth. The signaling cascades mediating this effect remain to be determined.
体外原代视网膜神经节细胞（RGC）培养模型广泛用于研究青光眼等疾病的发病机制。已知与培养底物的生物力学相互作用会影响核心细胞功能。然而，RGC 培养物通常生长在刚性塑料或玻璃基底上。我们假设软聚丙烯酰胺凝胶基底可能会改变原代培养的 RGC 的存活和神经突生长。从出生后（第 1-6 天）Wistar 大鼠的视网膜中分离原代培养物，在玻璃盖玻片或具有不同杨氏弹性模量（0.75、10 或 30 kPa）的聚丙烯酰胺（PA）凝胶基底上生长。基底用聚-L-赖氨酸和/或层粘连蛋白包被。用抗β-III-微管蛋白对 RGC 进行免疫染色。评估总神经突长度、生长锥形态、RGC 密度、线粒体形态和运输以及存活途径（Erk1/2、Akt、CREB）。与玻璃相比，E = 10 kPa 的 PA 凝胶基底使总神经突长度显著增加了 1.5 倍（p = 0.02）。在 30 kPa 的凝胶上，生长锥面积显著增大了 5.3 倍（p = 0.01）。与玻璃相比，PA 凝胶上神经突生长和 RGC 存活对基底涂层的存在更为重要（多聚-L-赖氨酸>层粘连蛋白）。线粒体形态和运动以及存活途径的激活在这四种底物之间没有显著差异。PA 凝胶基底显著增强了 RGC 的神经突生长。介导这种效应的信号级联仍有待确定。
