Accumulation of lipofuscin is curtailed by a calorie-restricted (CR) diet with aging. (A) Sciatic nerve sections from 18- and 38-month-old animals fed ad libitum (AL) or CR diets were stained with 4-[2-(6-dibutylamino)-2-naphthalenyl)ethenyl]-1-(3-sulfopropyl) hydroxide (di-8 ANEPPS) dye, which labels lipofuscin content (red). Large (∼10 μm) and smaller (<10 μm) di-8 ANEPPS-positive adducts are marked with arrows and arrowheads, respectively. Nuclei are labeled with Hoechst dye (blue). Scale bar, 20 μm. (B) Quantification of mean di-8 ANEPPS dye-positive pixels per fixed area is shown (*p < 0.05, ***p < 0.001, unpaired t-test, mean ± standard error of the mean [SEM], n = 3). (Color images available online at www.liebertonline.com/rej)

Age-related increase in proinflammatory mediators is attenuated by a lifelong calorie-restricted (CR) diet. (A) Total sciatic nerve lysates (20 μg/lane) from the indicated ages and diet were analyzed with antibodies against tumor necrosis factor-α (TNF-α, p50 and p65 subunits of NF-κB, and phospho-IκB. Tubulin is shown as a loading control. Molecular mass is shown on the left in kDa. (B) Quantification of TNF-α band intensities after normalization to tubulin is represented (^##p < 0.01, Fisher protected least significant difference (PLSD) analysis; *p < 0.05, unpaired t-test, mean ± standard error of the mean [SEM], n = 3). (C) Quantification of the p50 subunit of NF-κB band intensities normalized to tubulin is shown (^##p < 0.01, ^###p < 0.001, Fisher PLSD analysis; *p < 0.05, unpaired t-test, mean ± SEM, n = 3). (D) Quantification of the p65 subunit of NF-κB band intensities normalized to tubulin is shown (p = 0.0546, ^##p < 0.01, ^###p < 0.001, Fisher PLSD analysis; *p < 0.05, unpaired t-test, mean ± SEM, n = 3). (E) Phospho-IκB levels were also analyzed within the same lysates and quantification of band intensities normalized to tubulin was performed (p = 0.0830, ^##p < 0.01, ^###p < 0.001, Fisher PLSD analysis; **p < 0.01, unpaired t-test, mean ± SEM, n = 3). AL, Ad libitum.

A calorie-restricted (CR) diet minimizes accrual of damaged proteins within peripheral nerves during aging. (A) Western blot analysis of whole sciatic nerve lysates (20 μg/lane) from rats of the indicated months (mo) fed ad libitum (AL) or CR diets probed with an anti-ubiquitin antibody. Slow-migrating polyubiquitinated (pUbi) protein substrates are marked by a square bracket. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is shown as a protein loading control. Molecular mass in kDa is indicated on the left. (B) Quantification of pUbi band intensity normalized to GAPDH is shown (^#p < 0.05, ^###p < 0.001, Fisher protected least significant difference [PLSD] analysis; *p < 0.05, unpaired t-test, mean ± standard error of the mean [SEM], n = 3). In this and subsequent figures, the p value for Fisher protected least significant difference (PLSD) analysis is determined by comparing the 8-month-old sample with the older ages (18, 29, or 38 months) for each diet group. (C) Longitudinal sciatic nerve sections from 38-month-old animals fed ad libitum or CR diets were analyzed for ubiquitin- (red) and lysosomal associated membrane protein 1– (LAMP1) like (green) immunoreactivity. Arrows point to ubiquitin-positive protein aggregates adjacent to LAMP1-positive lysosomes (magnified 3 × in insets). Asterisks indicate intracytoplasmic vacuoles. Nuclei are labeled with Hoechst dye (blue). Scale bar, 20 μm.

Lipid peroxidation-associated modifications of proteins with age are relieved in sciatic nerve with a calorie-restricted (CR) diet. (A) Whole rat sciatic nerve lysates (20 μg/lane) were probed with a rabbit anti-malondialdehyde (MDA) antibody to detect MDA adducts to proteins (square bracket). Tubulin is shown as a protein loading control. (B) Quantification of MDA adduct band intensities normalized to tubulin is shown (^###p < 0.001, Fisher protected least significant difference (PLSD) analysis; *p < 0.05, unpaired t-test, mean ± SEM, n = 3). (C) Biochemical analysis of tyrosine residues (Nitros) on proteins (square bracket) using an anti-nitrosylation antibody in the same lysates as in A. Tubulin is shown as a protein loading control. (D) Quantification of nitrotyrosine band intensities from Western blots normalized to tubulin is shown (^##p < 0.01, Fisher's PLSD analysis; *p < 0.05, unpaired t-test, mean ± standard error of the mean [SEM], n = 3). (E) Western blot analysis of 4-hydroxynonenal (HNE) in the same lysates used in A and C. Tubulin is shown as a protein loading control. In A, C, and E, molecular mass is shown on the left in kDa. (F) Quantification of HNE adduct band intensities from western blots normalized to tubulin is shown (^###p < 0.001, Fisher PLSD analysis; p = 0.0697, unpaired t-test, mean ± SEM, n = 3). (G) Immunohistochemical staining of sciatic nerve from 18- and 38-month-old animals fed ad libitum (AL) and CR diets with an antibody against HNE. Nuclei are labeled with Hoechst dye (blue). Scale bar, 20 μm. (Color images available online at www.liebertonline.com/rej)

A calorie-restricted (CR) diet diminishes macrophage infiltration of peripheral nerves with age. (A) Cryosections of sciatic nerves from 18- and 38-month-old rats either maintained on ad libitum (AL) or CR diet were immunostained with an antibody against CD11b (green). Nuclei are stained with Hoechst dye (blue). Scale bar, 20 μm. (B) Quantification of CD11b⁺ cells per 0.1 mm² of nerve tissue area is shown for 18- and 38-month-old animals fed ad libitum or CR diets (***p < 0.001, unpaired t-test, mean ± standard error of the mean (SEM), n = 3). (C) Western blot analysis of sciatic nerve lysates (20 μg/lane) for steady-state levels of CD11b and endogenous immunoglobulin G (IgG) (heavy chain [HC] and light chain [LC]) from ad libitum and CR rats is shown. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is used as a protein loading control. Molecular mass in kDa is indicated on the left. (D) Quantification of the CD11b band intensities normalized to GAPDH from western blot analyses from whole sciatic nerve lysates is shown (^#p < 0.05, ^###p < 0.001, Fisher PLSD analysis; **p < 0.01, unpaired t-test, mean ± SEM, n = 3). (E) Quantification of the IgG-HC band intensities normalized to GAPDH from whole sciatic nerve lysates of ad libitum and CR rats of the indicated ages is shown (^##p < 0.01, Fisher PLSD analysis; *p < 0.05, unpaired t-test, mean ± SEM, n = 3). (F) Quantification of band intensities of IgG-LC normalized to GAPDH was performed (^##p < 0.01, ^###p < 0.001, Fisher PLSD analysis; *p < 0.05, unpaired t-test, mean ± SEM, n = 3). (Color images available online at www.liebertonline.com/rej)