Neurite outgrowth assays

The CNS of 20 individuals leeches of the same stage of development were dissected, microglia and neurons were separately collected as described above. Neurones were pooled in a homogeneous cell preparation to avoid individual polymorphism and in order to carry out a single, statistically relevant experiment. Neurons were evenly plated in 4-well Labtech culture chambers and were primarily cultured. After 6 days of culture, neurons were exposed to 10⁶ EVs/well from ODG EV-rich fractions (F4 to F6) issued from microglia isolated from the same individuals or to complete L-15 medium for negative control. Medium renewal, with or without EVs, was done every 4 days. Image acquisition on living cells was realised with a microscopy station Nikon Eclipse Ti2 (Nikon, Minato, Tokyo, Japan). This station, equipped with a perfect focus system, which automatically rectifies focus control for drift, is adapted for real time analysis. Images acquisitions were realised at days 6 and 20 of culture to quantify neurites outgrowth in EVs-stimulated vs. control neurons. Each well was totally scanned and a mosaic image was created for each condition. Manual measurement of the length of neurites was performed using ImageJ software only for individual neurons presenting neurites at both 6 and 20 days of culture. Measurements were done for each neuron, independently from the others, and are expressed as a percentage of growth compared to the first day of EVs exposure (= T6days), calculated as follow: [Percentage of growth = ((measure at 20 days*100)/measure at 6 days)/100]. They were then analysed with GraphPad software using a statistic “unpaired t test”.