Figure 1.
Reduction in peripheral nerve function in dbdb and Sod1−/− mice.
Electrophysiologic measurements were performed on dbdb and Sod1−/− mice with their corresponding littermate controls. All measurements of nerve conduction velocity (NCV) and latency were performed under isofluorane anesthesia with skin temperatures between 33°C and 34°C. Sciatic NCV was measured in (A) 5 month old dbdb and (B) 6 month old Sod1−/− mice. Tail distal motor latency was measured in (C) dbdb and (D) Sod1−/− mice. Statistically significant differences obtained from the different groups (n = 15) were tabulated as follows for the mean ± SEM (*p<0.05 by two-tailed t-test).
Figure 2.
Reductions in myelin and neuron structure in dbdb and Sod1−/− mice.
Thick sections of sciatic nerves were sectioned and visualized at 100x (A) non-diabetic dbm mouse and (B) dbdb mice. (C) Quantification of axon diameter/area, nerve fiber diameter/area, myelin area/thickness and g ratio (axon diameter/fiber diameter) were tabulated from thick sections of dbdb and control mice (n = 3). Thick sections of (D) 6-mo-old control and (E) 6-mo-old Sod1−/− mice are shown. (F) Quantification was performed similarly to that shown in 2C. Thick sections of (G) 20-mo-old control and (H) 20-mo-old Sod1−/− mice are presented. (I) Quantification was performed similarly to that in 2C and results expressed as mean ± SEM of n = 3 mice/group. Results were analyzed by two-tailed t-test (*p<0.05 by two-tailed t-test).
Figure 3.
Increase in sciatic nerve protein carbonyls in dbdb and Sod1−/− mice.
Total sciatic nerve cytosolic protein bound carbonyls in (A) dbdb and (B) Sod1−/− mice are presented. Total protein bound detergent-soluble protein carbonyls in (C) dbdb and (D) Sod1−/− mice are shown. Results are expressed as mean ± SEM (n = 6; *p<0.05 by two-tailed t-test).
Figure 4.
Increase in hydrophobic domain of protein exposure in dbdb and Sod1−/− mice.
Total BisANS labeling of sciatic nerve homogenates for (A) dbdb and (B) Sod1−/− mice are presented. Results are expressed as mean ± SEM (n = 6; *p<0.05 by two-tailed t-test).
Figure 5.
Determination of PMP22 protein carbonylation, changes in hydrophobicity, and aggregation in Dbdb mice.
(A) A Kyte-Doolittle Hydropathy plot was determined for PMP22 hydrophobicity. FTC labeled (B) cytosolic and (C) detergent-soluble protein fractions were immunoprecipitated with anti-PMP22 polyclonal antibody, loaded onto SDS-PAGE gels and analyzed for protein carbonylation. (D) The detergent soluble fraction was analyzed by Western blot against PMP22. The higher order PMP22 aggregates are indicated by *. Results are expressed as mean ± SEM (n = 6; *p<0.05 by two-tailed t-test).
Figure 6.
In vitro assessment of oxidative stress-induced aggregation of PMP22 protein.
Purified PMP22 protein was subjected to in vitro oxidation with increasing concentrations of tert-butyl hydroperoxide. SDS-PAGE was performed on oxidized PMP22 protein from both the soluble and detergent-soluble protein fractions. Results are expressed as mean ± SEM (n = 6; *p<0.05 by two-tailed t-test).