Neuronal Inflammation is Associated with Changes in Epidermal Innervation in High Fat Fed Mice

Abstract

Peripheral neuropathy (PN), a debilitating complication of diabetes, is associated with obesity and the metabolic syndrome in nondiabetic individuals. Evidence indicates that a high fat diet can induce signs of diabetic peripheral PN in mice but the pathogenesis of high fat diet-induced PN remains unknown. PURPOSE: Determine if neuronal inflammation is associated with the development of mechanical hypersensitivity and nerve fiber changes in high fat fed mice. METHODS: Male C57Bl/6 mice were randomized to a standard (Std, 15% kcal from fat) or high fat diet (HF, 54% kcal from fat) for 2, 4, or 8 weeks (n = 11-12 per group). Lumbar dorsal root ganglia were harvested and inflammatory mediators (IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, IL-12p70, IL-17, MCP-1, IFN-γ, TNF-α, MIP-1α, GMCSF, RANTES) were quantified. Hindpaw mechanical sensitivity was assessed using the von Frey test. Intraepidermal nerve fiber density (IENFD) and TrkA nerve fiber density were quantified via immunohistochemistry. RESULTS: After 8 weeks, HF had greater body mass (33.3 ± 1.0 vs 26.7 ± 0.5 g, p < 0.001), fasting blood glucose (160.3 ± 9.4 vs 138.5 ± 3.4 mg/dl, p < 0.05) and insulin (3.58 ± 0.46 vs 0.82 ± 0.14 ng/ml, p < 0.001) compared to Std. IL-1α, RANTES and IL-5 were higher in HF compared to Std after 2 and 4 weeks, respectively (IL-1α: 4.8 ± 1.3 vs 2.9 ± 0.6 pg/mg, p < 0.05; RANTES: 19.6 ± 2.2 vs 13.3 ± 1.2 pg/mg p < 0.05; IL-5: 5.8 ± 0.7 vs 3.1 ± 0.5 pg/mg, p < 0.05). IENFD and TrkA fiber density were also higher in HF vs Std after 4 weeks (IENFD: 39.4 ± 1.2 vs 32.2 ± 1.3 fibers/mm, p < 0.001; TrkA: 30.4 ± 1.8 vs 22.4 ± 1.3 fibers/mm). There were no significant differences in hindpaw sensitivity for Std vs HF. CONCLUSION: Increased inflammatory mediators preceded and accompanied an increase in cutaneous pain sensing nerve fibers in high fat fed mice but was not accompanied by significant mechanical allodynia. Diets high in fat may increase neuronal inflammation and lead to increased nociceptive nerve fiber density.

Keywords: TrkA; insulin resisitance; intraepidermal nerve fiber density; mechanical allodynia; peripheral neuropathy; prediabetes; von Frey.

FIGURE 1

Body Mass and Fasting Blood Glucose and Insulin Levels. HF exhibit greater body mass (A), fasting blood glucose (B), and insulin (C) levels compared to Std after 8 weeks of dietary intervention. ^a p < 0.05 vs wk 2 within diet, ^b p < 0.05 vs wk 4 within diet, and ^c p < 0.05 for Std vs HF within the week. Data are presented as mean ± SEM (n = 11–12 mice per group).

FIGURE 2

Hindpaw Mechanical Sensitivity. Data are presented as mean ± SEM (n = 11–12 mice per group). No significant differences.

FIGURE 3

Epidermal Innervation. Intraepidermal nerve fiber density (IENFD) includes all PGP 9.5 positive fibers that cross the epidermal-dermal border (A), TrkA nerve fiber density includes all TrkA positive fibers that cross the epidermal-dermal border (B), and TrkA: IENFD ratio (C). IENFD and TrkA nerve fibers peak and are greater than Std after 4 weeks of HF feeding. ^a p < 0.05 vs wk 2 within diet, ^b p < 0.05 vs wk 4 within diet, and ^c p < 0.05 for Std vs HF within the week. Data are presented as mean ± SEM (n = 8–12 mice per group).

FIGURE 4

Representative Images of Epidermal Innervation. Fluorescent images show nerve fibers for Std (top panel) and HF (lower panel) after 8 weeks of dieteray intervention. Nerve fibers that crossed the epidermal/dermal border were quantified and expressed as fibers per mm. Anti-PGP 9.5 (red) labeled all nerve fibers and anti-TrkA (green) labeled TrkA nerve fibers.

FIGURE 5

Inflammatory Mediators in the Lumbar DRG. Std vs HF after 2 weeks (A), 4 weeks (B), and 8 weeks (C) of dietary intervention. Data are presented as mean ± SEM (n = 3–12 mice per group). ^* p < 0.05 vs for Std vs HF. IL-10, IFN-γ, TNF-α, MIP-1a, GMCSF were below the limits of detection in the majority of the samples, thus means could not be calculated.

FIGURE 6

Inflammatory Mediators in the Lumbar DRG in HF Group after 2, 4, and 8 Weeks. Data are presented as mean ± SEM (n = 3–11 mice per group). ^a p < 0.05 vs wk 2, ^b p < 0.05 vs wk 4. IL-10, IFN-γ, TNF-α, MIP-1α were below the limit of detection in the majority of the samples, thus means could not be calculated.