Ease in the overall level of protein carbonyls in theProtein Oxidation, Misfolding and DemyelinationFigure 2. Reductions in myelin and neuron structure in dbdb and Sod12/2 mice. Thick sections of sciatic nerves were sectioned and visualized at 100x (A) non-diabetic dbm mouse and (B) dbdb mice. (C) Title Loaded From File 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-moold control and (E) 6-mo-old Sod12/2 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 Sod12/2 mice are presented. (I) Quantification was performed similarly to that in 2C and results expressed as mean 6 SEM of n = 3 mice/group. Results were analyzed by two-tailed t-test (*p,0.05 by two-tailed t-test). doi:10.1371/journal.pone.0065725.gDiscussionIn humans, the diabetic etiology includes neuronal dysfunction, axonal degeneration, Schwann cell dysfunction, and chronic motor/sensory neuron demyelination with increasing disease severity [1]. It has been reported that Schwann cell death [45], neuronal cell death as observed in streptozotocin-induced diabetes [46], and damage to motor/sensory neurons [46] are associated with diabetic neuropathy and reduced nerve function. All of these changes are likely contributors to loss of muscle mass, limb pain and dysfunction of the lower extremities in dbdb mouse model of peripheral neuropathy. Chronic oxidative stress is believed to be one of the leading underlying mechanisms of reduced myelination in diabetic neuropathy. Indeed, recent studies have shown that reduction in oxidative stress may improve sciatic nerve/myelin morphology and function in diabetic neuropathy [4,5]. It has also been well Title Loaded From File documented that oxidative stress plays a key role in initiating misfolding and aggregation of proteins and enzymes[36,41,42,43,44,47]. However, it is unknown whether imbalanced structural states of proteins (protein oxidation, misfolding and aggregation) are linked to reduced nerve conduction and myelin thickness in peripheral neuropathies, in part, due to the lack of sensitive technologies to measure these changes. In this study, we report for the first time that sciatic nerve proteins of dbdb and Sod12/2 mice undergo oxidation (protein carbonylation) and alteration in protein conformation which may be closely linked to decrement in nerve conduction and altered myelin morphology. The novelty of this study is the use of two mouse models to determine the role of oxidative stress in peripheral neuropathy (i) the well-characterized dbdb mouse model of diabetic peripheral neuropathy that is known to be associated with oxidative stress/ damage [2,24] and (ii) the Sod12/2 mouse model of in vivo oxidative stress which exhibits age-associated deficits in neuromuscular function [25,26,27,28]. We found significant changes in protein carbonylation and misfolding in diabetic mice which correlated well with reductions in sciatic NCV and myelinProtein Oxidation, Misfolding and DemyelinationFigure 3. Increase in sciatic nerve protein carbonyls in dbdb and Sod12/2 mice. Total sciatic nerve cytosolic protein bound carbonyls in (A) dbdb and (B) Sod12/2 mice are presented. Total protein bound detergent-soluble protein carbonyls in (C) dbdb and (D) Sod12/2 mice are shown. Results are expressed as mean 6 SEM (n = 6; *p,0.05 by t.Ease in the overall level of protein carbonyls in theProtein Oxidation, Misfolding and DemyelinationFigure 2. Reductions in myelin and neuron structure in dbdb and Sod12/2 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-moold control and (E) 6-mo-old Sod12/2 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 Sod12/2 mice are presented. (I) Quantification was performed similarly to that in 2C and results expressed as mean 6 SEM of n = 3 mice/group. Results were analyzed by two-tailed t-test (*p,0.05 by two-tailed t-test). doi:10.1371/journal.pone.0065725.gDiscussionIn humans, the diabetic etiology includes neuronal dysfunction, axonal degeneration, Schwann cell dysfunction, and chronic motor/sensory neuron demyelination with increasing disease severity [1]. It has been reported that Schwann cell death [45], neuronal cell death as observed in streptozotocin-induced diabetes [46], and damage to motor/sensory neurons [46] are associated with diabetic neuropathy and reduced nerve function. All of these changes are likely contributors to loss of muscle mass, limb pain and dysfunction of the lower extremities in dbdb mouse model of peripheral neuropathy. Chronic oxidative stress is believed to be one of the leading underlying mechanisms of reduced myelination in diabetic neuropathy. Indeed, recent studies have shown that reduction in oxidative stress may improve sciatic nerve/myelin morphology and function in diabetic neuropathy [4,5]. It has also been well documented that oxidative stress plays a key role in initiating misfolding and aggregation of proteins and enzymes[36,41,42,43,44,47]. However, it is unknown whether imbalanced structural states of proteins (protein oxidation, misfolding and aggregation) are linked to reduced nerve conduction and myelin thickness in peripheral neuropathies, in part, due to the lack of sensitive technologies to measure these changes. In this study, we report for the first time that sciatic nerve proteins of dbdb and Sod12/2 mice undergo oxidation (protein carbonylation) and alteration in protein conformation which may be closely linked to decrement in nerve conduction and altered myelin morphology. The novelty of this study is the use of two mouse models to determine the role of oxidative stress in peripheral neuropathy (i) the well-characterized dbdb mouse model of diabetic peripheral neuropathy that is known to be associated with oxidative stress/ damage [2,24] and (ii) the Sod12/2 mouse model of in vivo oxidative stress which exhibits age-associated deficits in neuromuscular function [25,26,27,28]. We found significant changes in protein carbonylation and misfolding in diabetic mice which correlated well with reductions in sciatic NCV and myelinProtein Oxidation, Misfolding and DemyelinationFigure 3. Increase in sciatic nerve protein carbonyls in dbdb and Sod12/2 mice. Total sciatic nerve cytosolic protein bound carbonyls in (A) dbdb and (B) Sod12/2 mice are presented. Total protein bound detergent-soluble protein carbonyls in (C) dbdb and (D) Sod12/2 mice are shown. Results are expressed as mean 6 SEM (n = 6; *p,0.05 by t.
