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. 2022 Nov 25;13:7269. doi: 10.1038/s41467-022-34846-w

Fig. 7. Negr1 exerts a trophic effect on sympathetic neurons.

Fig. 7

a Immunofluoescence images showing Negr1 and TYR.H. distribution in wt and ob/ob TH PVAT sections (from 1 out of 2 replicates). Insets show higher magnification of framed areas. Nuclei are counterstained with DAPI. Scale bar: 50 µm. b Representative micrographs of SCG sympathetic neurons cultured at low density for 24 h in presence of 1 or 10 ng/ml NGF, with or without 500 ng/ml recombinant Negr1, and stained for β3-tubulin. Scale bar: 100 µm. c Quantifications of total axonal length, branching points and principal neurites on primary sympathetic neurons incubated for 24 h in medium supplemented with the indicated factors. Violin plots show median and quartiles of 80 < N < 120 neurons per condition from 3 independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 by one-way ANOVA followed by Tuckey’s multiple comparisons test. d Bar chart shows the quantification of axonal swellings normalised on total axonal length. Data are shown as mean ± SEM of 40 to 80 random fields from 3 independent experiments analysed by one-way ANOVA followed by Tuckey’s multiple comparison test. ****p < 0.0001. e Representative live BF images of SCG sympathetic neurons seeded on microfluidic devices, with the axonal chamber incubated for 24 h with conditioned media (C.M.) from wt and ob/ob fat explants +/– recombinant Negr1 (500 ng/ml). Arrows point at axonal swellings. Scale bars: 20 µm. f Localisation of catheter-connected osmotic minipumps implanted subcutaneously in ob/ob mice. Created with BioRender.com. g Trend of ob/ob mice weight from the surgery day until mice were culled. h Bar graph showing the weight of SC WAT pads on day 14 after minipumps implantation. i Western blot showing TYR.H. and β3-TUB. abundances in the SC WAT of ob/ob mice implanted with subcutaneous minipumps delivering either saline (vehicle) or recombinant Negr1 and relative quantification. N = 5 biological replicates, unpaired two-tailed independent t-test (unequal distribution). Data are shown as mean ± SEM. TH thoracic PVAT, NGF nerve growth factor, Negr1 neuronal growth regulator 1, TYR.H. tyrosine hydroxylase, β3-TUB β3-tubulin, SCG superior cervical ganglia, M molecular weight marker. Source data are provided as a Source Data file.