Abstract
Purpose :
Directional outgrowth of neurites in primary cultures of retinal ganglion cells (RGC) would facilitate neurite-specific analyses, such as active transport or response to mechanical stress. Unlike many neuron populations, RGC primary cultures do not grow well in microfluidic chambers. We investigated methods of guiding RGC neurites with microchannels on elastic polyacrylamide (PA) hydrogels and scratches on glass coverslips without application of fluid flow.
Methods :
P2-12 rat retinae were used to set up primary cultures of retinal cells. Neurite outgrowth was enhanced by baculovirus-mediated transduction with hyper-IL6. All culture surfaces were poly-l-lysine and laminin coated. RGC were identified by beta-III-tubulin immunostaining. Using polypropylene surgical sutures, open (roofless) microchannels were created in an elastic PA gel matrix (Young’s modulus E = 30 kPa) and cells were seeded into the channels. Channel width was varied in a range from 30 to 100 µm. In a second approach linear scratches were made into glass coverslips. RGC number and neurite morphology (growth direction and straightness index) were assessed at day 4 using ImageJ software.
Results :
Good RGC survival was only achieved in 100 µm channels (RGC number per field of view: 100µm 4.9±2.5, 50µm 0.7±0.6 (p=0.001), 30µm 0.5±0.5 (p=0.0002). The direction of neurite outgrowth within channels was mainly longitudinal or perpendicular (50:50) to the channel axis (78%; n=24). Oblique outgrowth occurred in 22% of RGC. Compared to the central channel groove, the channel rim was a better guiding structure. Glass scratches guided neurites even more tightly (std. deviation of deviation angle from the guiding structure: channel rim 20.2°, scratch 1.7°; p<0.0001 (F test). Guided neurites grew straighter than neurites on plain, non-guiding, surfaces (30kPa PA gel: p=0.03, glass: p<0.0001). The straightness index was not different between scratches and channel rims (p=0.1).
Conclusions :
In the absence of flow, topographically guided neurite outgrowth was obtained with both, gel channels and glass scratches. Guiding was most efficient with glass scratches. The biophysical mechanisms underlying topographical guidance of RGC neurites remain to be investigated.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.