Figure 4.

Cytoplasmic SRF facilitates facial nerve regeneration in vivo. A, A¹, The facial nerve is outlined in blue. A², Ad-GFP or Ad-SRF-ΔNLS-GFP virus (green) was injected. The injection and axotomy position is depicted by an arrow. A³, Tracer (FG, red) is injected in the whisker pad and retrogradely transported to cell bodies of regeneration or sprout forming neurons. A⁴, Lesioned facial nucleus of an Ad-SRF-ΔNLS-GFP-injected animal. Overexpression of SRF-ΔNLS-GFP resulted in cytoplasmic SRF localization. A⁵, Higher magnification of neuron labeled by asterisk in A⁴. B–E, Inspection of axon regeneration of GFP expressing neurons only. The number of FG-positive neurons on the unlesioned side was comparable between both virus types (B, D). In Ad-GFP-infected animals, ∼30% of all neurons expressing GFP were also tracer positive (labeled with an arrow in C). In contrast, >70% of SRF-ΔNLS-GFP-expressing motoneurons were positive for the regeneration, indicating tracer (labeled with arrows in E). F–I, Visualization of the total population of FG-positive motoneurons in control (F, H) or lesioned (G, I) facial nuclei of GFP- (F, G) or SRF-ΔNLS-GFP (H, I)-expressing animals. SRF-ΔNLS-GFP enhanced the number of tracer-positive neurons in lesioned facial nucleus (I) compared with GFP (G). J–M, Nissl staining labels all facial motoneurons. In Ad-GFP-infected animals, the number of motoneurons was decreased upon lesion (J, K). In contrast, SRF-ΔNLS-GFP prevented motoneuron loss upon facial nerve injury (L, M). N–Q, In Ad-GFP-expressing facial nuclei, galanin-positive neurons were downregulated upon lesion (N, O). In contrast, more galanin-positive neurons were present upon lesion in animals expressing SRF-ΔNLS-GFP (P, Q). R, Quantification of axonal regeneration counting all FG tracer-positive cells (each square depicts one animal). The average number of FG-positive neurons/section upon lesion is increased by SRF-ΔNLS-GFP. S, Percentage of tracer-positive neurons in the total motoneuron population depicted by the ratio of neurons present on the control and lesioned side. T, U, SRF-ΔNLS-GFP increased the number of Nissl- (T) and galanin-positive (U) neurons upon facial nerve injury. Scale bars: A⁴, F–Q, 100 μm; B–E, 10 μm; A⁵, = 5 μm.