Oxidative injury and neuropathy in diabetes and impaired glucose tolerance

Abstract

Clinical studies suggest that impaired glucose tolerance (IGT) is associated with the development of neuropathy. The aim of the current study was to determine if neuropathy developed in the female Zucker Diabetic Fatty (ZDF) rat, an animal model of IGT and type 2 diabetes. The ZDF rat develops impaired glucose tolerance (IGT) when fed a control diet, and frank diabetes when fed a high fat diet. Following 10 weeks of hyperglycemia, sensory nerve action potentials (SNAP) and compound motor action potentials (CMAP) were reduced and sensory conduction velocities were slowed (distal>proximal) in the tail and hind limb in ZDF animals with IGT and frank diabetes (p<0.01). Neuropathy was coupled with evidence of increased reactive oxygen species (ROS) and cellular injury in dorsal root ganglion (DRG) neurons from IGT animals. Our study supports the hypothesis that neuropathy develops in an animal model of IGT and is associated with evidence of oxidative injury in DRG and peripheral nerves.

Fig 1. Female ZDF rats develop obesity and hyperglycemia

Female ZDF-HF or ZDF-N animals become progressively obese. Lean control animals do not become obese (A). ZDF-HF females develop diabetes. In contrast, ZDF-N animals develop IGT, but are not continuously hyperglycemic. Controls remain normoglycemic (B). IGT is observed in ZDF females after glucose tolerance testing (C). The blood glucose peaked at 30 min, rising to 820 mg/dl in ZDF-HF, and 444 mg/dl in ZDF-N animals. In contrast, glucose levels remained normal after a GTT in control animals.

Fig 2. Level of hyperglycemia in ZDF rats is associated with reduction in sensory nerve action potentials and conduction velocities

Mean tail and first digit SNAP amplitudes are severely reduced in diabetic (ZDF-HF) and IGT (ZDF-N) animals (A). SNAP amplitudes obtained from the distal tail are more severely reduced than digit SNAP amplitudes, consistent with a length dependent axonal neuropathy. Amplitudes are smaller in ZDF-HF than ZDF-N animals. Tail and digit mean sensory conduction velocities (SCVs) are more severely reduced in ZDF-HF than ZDF-N animals (B). Control animals had normal NCS. * = p<0.05, ** = p<0.01 compared to lean control.

Fig 3. Hyperglycemia in ZDF rats reduces motor action potential responses

The tail and sciatic/peroneal CMAP amplitudes (A), sciatic/peroneal MCVs and tail MCVs (B) are severely reduced, and tail DML prolonged in both ZDF-HF and ZDF-N animals, consistent with a length dependent process. There was also no significant difference in the hind limb F-wave responses in diabetic animals, consistent with preserved motor axons in proximal nerves. * = p<0.05, ** = p<0.01 *** = p<0.001 compared to lean control.

Fig 4. Markers of oxidative stress in DRG neurons from lean control ZDF-HF and ZDF-N animals

Levels of MDA (A–C and G), or HNE (D–F and H) were measured in over 100 DRG neurons in each animal. Levels of MDA and HNE were increased in both IGT as well as diabetic animals, consistent with evidence of early oxidative injury associated with hyperglycemia. * = p<0.05, ** = p<0.01 *** = p<0.001 compared to lean control.

Fig 5. Caspase-3 cleavage is increased in DRG and SC from ZDF-HF and ZDF-N rats

Caspase cleavage in DRG sections (A). The upper row of panels indicates DRG DIC images from: lean control animals on a high fat diet (i), diabetic animals (ZDFHF) (ii), IGT animals (ZDFN) (iii). The lower panel shows the corresponding DRG sections stained for cleaved (active) caspase-3 (red), and nuclear chromatin (blue), indicated by white arrow heads (see inset in middle panel). There is no caspase-3 staining in (i), but increased cleaved caspase-3 in the cytoplasm (white arrows) in neurons from diabetic (ii) compared to IGT neurons (iii). Caspase-3 cleavage (active caspase-3) is measured in DRG neurons (B) and SC (C). ** = p<0.01 compared to lean control.

Fig 6. Apoptotic changes are increased in DRG from ZDF-HF and ZDF-N rats

DRG nuclei were stained using the TUNEL technique (A). The figure represents composite immunohistochemical staining overlaying the DIC image. i–iii: Low magnification images. iv–vi: High magnification images. i and iv: TUNEL positive neurons are rare in control sections. ii and v: DRG from diabetic animals (ZDF-HF) showing numerous apoptotic neuronal nuclei (arrow), interspersed with non-apoptotic neurons (TUNEL staining negative) consistent with PCD in single cells. iii and vi. DRG from IGT animals (ZDF-N) showing apoptotic nuclei (arrows). TUNEL staining is measured in DRG neurons (B) and SC (C). ** = p<0.01 *** = p<0.001 compared to lean control.