Impaired sensory nerve function and axon morphology in mice with diabetic neuropathy

Abstract

Diabetes is the most prevalent metabolic disorder in the United States, and between 50% and 70% of diabetic patients suffer from diabetes-induced neuropathy. Yet our current knowledge of the functional changes in sensory nerves and their distal terminals caused by diabetes is limited. Here, we set out to investigate the functional and morphological consequences of diabetes on specific subtypes of cutaneous sensory nerves in mice. Diabetes was induced in C57Bl/6 mice by a single intraperitoneal injection of streptozotocin. After 6-8 wk, mice were characterized for behavioral sensitivity to mechanical and heat stimuli followed by analysis of sensory function using teased nerve fiber recordings and histological assessment of nerve fiber morphology. Diabetes produced severe functional impairment of C-fibers and rapidly adapting Aβ-fibers, leading to behavioral hyposensitivity to both mechanical and heat stimuli. Electron microscopy images showed that diabetic nerves have axoplasm with more concentrated organelles and frequent axon-myelin separations compared with control nerves. These changes were restricted to the distal nerve segments nearing their innervation territory. Furthermore, the relative proportion of Aβ-fibers was reduced in diabetic skin-nerve preparations compared with nondiabetic control mice. These data identify significant deficits in sensory nerve terminal function that are associated with distal fiber loss, morphological damage, and behavioral hyposensitivity in diabetic C57Bl/6 mice. These findings suggest that diabetes damages sensory nerves, leading to functional deficits in sensory signaling that underlie the loss of tactile acuity and pain sensation associated with insensate diabetic neuropathy.

Figures

Fig. 1.

Streptozotocin (STZ)-treated mice develop diabetes, weight loss, and sensory hypoalgesia over the course of 8 wk. A: average blood glucose level of diabetic (STZ-injected) and nondiabetic (vehicle-injected) mice. B: average weight of diabetic and nondiabetic mice. C: behavioral mechanical response threshold (von Frey filament applied to the plantar hindpaw). D: behavioral thermal response latency (stimulus applied to the plantar hindpaw). **P < 0.01 by Student's t-test; ††P < 0.01 by Wilcoxon rank-sum test.

Fig. 2.

Diabetic nerves appear mostly similar to nondiabetic nerves at low magnification. A: plastic-embedded 0.5-μm sections of saphenous nerve stained with toluidine blue and imaged by oil immersion light microscopy (×800 magnification). Top, proximal nerve segments from nondiabetic (left) and diabetic mice (right) taken from above the knee; bottom, distal, subdermal nerve segments from nondiabetic (left) and diabetic mice (right) taken from the dorsal hindpaw. Myelinated axons from diabetic mice appeared marginally less circular than axons from nondiabetic mice. Note the separations between the axon and myelin sheath in the distal diabetic nerve (highlighted by arrows). B: myelinated axon counts from nondiabetic and diabetic proximal nerve segments (P > 0.05 by Wilcoxon rank-sum test). C: myelinated axon counts from distal nerve segments (P > 0.05 by Wilcoxon rank-sum test).

Fig. 3.

Diabetic nerves exhibit morphological changes at high magnification. A: high-magnification image of nondiabetic axons in a distal nerve segment stained with uranyl acetate and lead citrate (magnification: ×8,710). The central myelinated axon within the size range of a sensory Aβ-fiber (6–12 μm) exhibited a single mitochondrion, a lightly stained neurofilament network, and some more intensely stained microtubules. Smaller myelinated axons within the size range of sensory Aδ-fibers (1–5 μm) exhibited few mitochondria and a similar filament network. B: high-magnification image of diabetic axons in a distal nerve segment (magnification: ×9,260). Both axons fell within the size range of sensory Aβ-fibers. Both axons exhibited an increased number of mitochondria (arrows) and a dense neurofilament network compared with nondiabetic axons. The top right of the left axon demonstrated a widened interface between the Schwann cell and the axoplasm (arrowheads). The right axon demonstrated a similar area that was partly extracted (arrowheads).

Fig. 4.

Axon-myelin separations are prevalent in distal axons from diabetic mice. A: electron micrographs of myelinated and unmyelinated axons in distal nerve segments from nondiabetic (top) and diabetic mice (bottom) stained with uranyl acetate and lead citrate (×3,430 magnification). Myelinated axons exhibited axon-myelin separations and numerous intracellular organelles compared with nondiabetic axons. Arrows highlight lipid-like material within axon-myelin separations. Arrowheads highlight some unmyelinated axons, which appeared more electron dense in diabetic nerves. B: frequency of axon-myelin separation in proximal versus distal nerve segments in nondiabetic and diabetic mice. C: frequency of axon-myelin separation in small-diameter axons (lower quartile) versus large-diameter axons (upper quartile). Quartiles were defined from the size distribution of all myelinated axons in nondiabetic or diabetic samples. †††P < 0.001 by a Kruskal-Wallis test followed by a post hoc Dunns multiple-comparison test between selected groups; §P < 0.05 by a Mantel-Haenszel test of homogeneity.

Fig. 5.

Diabetic mice preferentially lose functional Aβ-fiber innervation. A: proportion of functional Aβ-, Aδ-, and C-fibers identified in diabetic and nondiabetic skin-nerve preparations. B: proportion of functional Aβ-fibers that were rapidly adapting (RA) versus slowly adapting (SA) in diabetic and nondiabetic skin-nerve preparations. C: proportion of functional Aδ fibers that were rapidly adapting (D hair) versus slowly adapting (AM) in diabetic and nondiabetic skin-nerve preparations. ‡‡‡P < 0.001 by a χ2-test followed by analysis of standardized residuals.

Fig. 6.

Diabetes impairs sensory nerve fiber function. A–E: force stimulus response curves in C-fibers (5–200 mN, 10 s each, 1-min interstimulus interval; A) and SA Aβ-fiber responses (B), RA Aβ-fiber responses (C), AM fiber responses (D), and D-hair fiber responses (E) to increasing mechanical stimuli in diabetic versus nondiabetic mice. AP, action potential. *P < 0.05 and **P < 0.01 by two-way ANOVA followed by a post hoc Bonferroni t-test.

Fig. 7.

Diabetes increases heat response thresholds in C-fibers. A: proportion of C-fibers sensitive to heat. B: numbers of APs elicited by heat ramp (32 to 52°C over 10 s). C: heat response threshold. D: proportion of C-fibers sensitive to 1 μM capsaicin. E: numbers of APs elicited by 1 μM capsaicin. †P < 0.05 by a Wilcoxon rank-sum test.