Fig 2: Aggregation of human IAPP is required to induce neuropathic pain.(A and B) Fluorescence (indicates amount of amyloid fibrils) of thioflavin T (A) and transmission electron microscopy imaging (scale bars: 0.2 nm) (B) after 24 hours of incubation of hIAPP, mIAPP, or pramlintide. (C) Sensory neurons were treated with 100 nM hIAPP, mIAPP, pramlintide, or saline for 24 hours. The average neurite length per neuron was assessed and expressed as the percentage length per neuron of vehicle-treated neurons (n = 8; n represents a DRG culture of 1 mouse). (D) Mitochondrial ROS level in cultured DRG neurons incubated with hIAPP or pramlintide (10, 100, and 1,000 nM). Measurements are per cell from 3 different cultures, n = 551–654 neurons per group. (E and F) Course of mechanical sensitivity (n = 5) (E) and IENF density (n = 7) on day 6 (F) after intraplantar injection of 1,000 fg hIAPP, mIAPP, and pramlintide in male and female WT mice. (G) Density of IENFs in skin of T2DM subjects (n = 6) and non-T2DM controls (n = 9). (H) Representative images of G stained for the pan-neuronal marker PGP9.5 and collagen IV (lines indicate the border between dermis and epidermis; white arrows represent the IENF; scale bar: 20 µm). (I) IAPP-positive oligomers in skin of T2DM subjects (n = 6) and non-T2DM controls (n = 9). (J) Representative images of I stained for collagen IV (C IV), IAPP, and oligomers (I11). IAPP- and oligomer-positive spots are indicated by arrowheads. Lines indicate the border between dermis and epidermis. (C and D) One-way ANOVA with Dunnett’s test; *P < 0.05, **P < 0.01, ***P < 0.001. (E and F) Two-way ANOVA with Tukey’s test; *P < 0.05 vs. vehicle; ##P < 0.01 vs. pramlintide. (G and I) Unpaired t test; *P < 0.05, **P < 0.01. Data are expressed as mean ± SEM.

Fig 3: hIAPP Ob/Ob mice have features of T2DM neuropathy.(A) Nonfasting blood glucose levels in WT (n = 8) and hIAPP Ob/Ob (n = 5–8) mice; 2-way ANOVA with Šidák’s test, ***P < 0.001. (B) Mechanical threshold of the plantar surface of WT (n = 8) and hIAPP Ob/Ob (n = 7) mice; 2-way ANOVA with Šidák’s test, ***P < 0.001. (C) Number of nerve fibers crossing from dermis to epidermis in WT (n = 8) and hIAPP Ob/Ob (n = 5) mice at age 15 weeks; unpaired t test, ***P < 0.001. (D) Representative images of paw skin of WT and hIAPP Ob/Ob mice stained for the pan-neuronal marker PGP9.5 and collagen IV (lines indicate the border between dermis and epidermis; white arrows represent IENF; scale bars: 20 µm). All experiments were performed in male mice. Data are expressed as mean ± SEM.