The authors have declared that no competing interests exist.
Conceived and designed the experiments: JC. Performed the experiments: FX HF J-FH. Analyzed the data: FX HF. Contributed reagents/materials/analysis tools: FX HF KJ. Wrote the paper: JC MC.
To establish the role of the metabolic state in the pathogenesis of polyneuropathy, an age- and sex-matched, longitudinal study in rats fed high-fat and high-sucrose diets (HFSD) or high-fat, high-sucrose and high-salt diets (HFSSD) relative to controls was performed. Time courses of body weight, systolic blood pressure, fasting plasma glucose (FPG), insulin, free fatty acids (FFA), homeostasis model assessment-insulin resistance index (HOMA-IR), thermal and mechanical sensitivity and motor coordination were measured in parallel. Finally, large and small myelinated fibers (LMF, SMF) as well as unmyelinated fibers (UMF) in the sciatic nerves and ascending fibers in the spinal dorsal column were quantitatively assessed under electron microscopy. The results showed that early metabolic syndrome (hyperinsulinemia, dyslipidemia, and hypertension) and prediabetic conditions (impaired fasting glucose) could be induced by high energy diet, and these animals later developed painful polyneuropathy characterized by myelin breakdown and LMF loss in both peripheral and central nervous system. In contrast SMF and UMF in the sciatic nerves were changed little, in the same animals. Therefore the phenomenon that high energy diets induce bilateral mechanical, but not thermal, pain hypersensitivity is reflected by severe damage to LMF, but mild damage to SMF and UMF. Moreover, dietary sodium (high-salt) deteriorates the neuropathic pathological process induced by high energy diets, but paradoxically high salt consumption, may reduce, at least temporarily, chronic pain perception in these animals.
International Diabetes Federation Diabetes Atlas estimates that by 2011, 366 million people worldwide have diabetes (accounting for 8.3% of adults) and diabetic patients are expected to increase to 552 million people by 2030
Peripheral neuropathy, with clinical manifestations of pain, sensory, autonomic and even motor dysfunctions, is a major common complication of both diabetes and prediabetes
Diabetic peripheral neuropathy (DPN) has been generally classified into two subgroups: (1) Typical DPN is a chronic, symmetrical sensorimotor polyneuropathy that is thought to be caused by long-standing hyperglycemia
The experiments were performed on three-month-old male Sprague-Dawley albino rats (purchased from Laboratory Animal Center of Fourth Military Medical University, FMMU, Xi'an, P.R. China). The animals were housed in plastic cages with access to food and water
The experimental protocols were approved by Institutional Animal Care and Use Committee of FMMU (Permit number: SCXK2007-007). The present study was performed in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23) revised 1996.
Thirty rats were randomly divided into three groups: one control group fed a conventional diet (CD, n = 10); the two treatment groups were fed HFSD (n = 10) and HFSSD (n = 10), respectively. The experiment lasted from post diet day (PDD) 1 to 120. The composition of the CD, HFSD and HFSSD and the mass or energy proportion of carbohydrate, protein and fat in the three groups are shown in supplementary data (
SBP was measured by a computerized multi-channel physio-recording and analytical system (RM-6280, Chengdu Medical Instrument Factory, P.R.China) every 5 days. Rats were measured five times on a fixation time (8:00–10:00) and averaged values were used as mean SBP.
All animals underwent a 12 hours overnight fasting before blood collected. Blood was collected every 5 days until PDD 30 by shearing tail tip between 8:00 and 9:00. Blood collection was practiced again at the end of the experiment. FPG was measured with One Touch blood glucose meter (Lifescan Inc., California, U.S.A.) directly. Plasma FFA was measured according to the protocol described by the manufacturer of FFA kit (Applygen Technologies Inc., Beijing, P.R.China). The fasting plasma insulin concentration was also measured according to the protocol described by the manufacturer of 125I-Insulin Radioimmunoassay Kit (Chemclin Biotech Co., Ltd., Beijing, P.R.China).
HOMA model, which incorporates measures of both FPG and insulin levels, was used as an index of IR and calculated with the following formula: [insulin ( µU/ml)×glucose (nM)÷22.5] as described previously
Somatic pain sensitivity was evaluated as an index of somatic sensory functions. Paw withdrawal mechanical threshold (PWMT) was measured using ascending graded individual von Frey monofilaments with bending forces of 3.5, 4.5, 5.5, 7.8, 11, 15, 20, 25, 30, 40, 50, 60 g as reported previously
Motor coordinating performance was tested using a Rota-rod treadmill (Ugo, Ltd., Italy). The rats were tested simultaneously on the apparatus with a rod-rotating speed of 6 rpm. The accelerating speed of the Rota-rod was set to increase from 6 rpm to 30 rpm within 2 min. The animals were placed on the treadmill and the timers were started with acceleration and automatically stopped when the animal fell off, with a maximal cutoff time of 300 s.
At the end of the whole experiment, three rats per group were infused with 2.5% glutaraldehyde and 4% paraformaldehyde in 0.1 M phosphate buffer (pH = 7.4) after anesthetization with sodium pentobarbital (80 mg/Kg; Sigma, Ltd., U.S.A). Then the sciatic nerves and spinal dorsal column were collected and postfixed by a fixative of 3% glutaraldehyde in 0.1 M phosphate buffer (pH = 7.4), overnight at 4 °C. Transverse sections (1 mm) of the spinal cord were obtained by vibrating microtome DTK-1000 (Dosaka, Ltd., Japan). The dorsal column were dissected out and cut into small pieces of similar dimensions, followed by osmification in 1% OsO4 in 0.1 M sodium cacodylate buffer for 2 hours at room temperature, dehydration in an ascending acetone series. The small-cutting-blocks of sciatic nerve were prepared in the same osmification and dehydration procedure. The osmicated tissue blocks were further embedded in Epon-812 (Serva, Ltd., Germany) and trimmed carefully under the light microscope. Ultrathin sections (50–70 nm) were cut perpendicularly to the axis of nerve fibers with a diamond knife on ultramicrotome LKB-11800 (LKB Ltd., Sweden) and collected by copper grids (300 meshes). The ultrathin sections stained with uranyl acetate and lead citrate were observed and microphotographs were taken under an electron microscope (EM, JEM-2000EX, JEOL Ltd., Japan).
When electron photomicrographs were taken, the areas of each axon and fiber profile were measured using Image Pro Plus 6.0, and the area of each fiber myelin profile was obtained based upon the formula: area of myelin profile = area of fiber profile–area of axon profile. The profile ratio was referred to as a ratio between the area of an axon profile and the area of a fiber profile according to the formula of the conventional G-ratio. However, because G-ratio is obtained by a ratio between circumference of an axon and circumference of a fiber (2πR of an axon/2πR of a fiber = radius of an axon/radius of a fiber) and the radius of the damaged fibers would produce great bias due to irregularity of the fiber shapes, it was not adopted in the current study.
The damaged fibers were also classified into four grades according to the intensity and extensity of destruction of myelinated and UMF axons for both the SN and the SDC (
Shapiro-Wilk normality test was first used to determine which data are normally or non-normally distributed. Normally distributed data were shown as mean±SEM, while non-normally distributed data were shown as median with maximum and minimum. Parametric one-way ANOVA followed by Fisher's PLSD post hoc analysis was used for comparisons of the normally distributed data (body weight, SBP, biochemistry parameters, and somatic sensorimotor behaviors) between CD and HFSD and HFSSD groups. Non-parametric Kruskal-Wallis H test and Mann-Whitney U test were used for comparisons of non-normally distributed data (areas of myelin profiles, profile ratios, and proportions of the damaged nerve fibers) between CD and HFSD and HFSSD groups. Value p<0.05 was considered to be significantly different.
The average foodstuff consumption per rat per day was measured across the whole time course from PDD 0 to 120. Rats fed CD diet consumed more foodstuff (35.3±0.3 g/day, n≥6) than either of the rats fed HFSD (29.2±0.2 g/day, n≥5) or HFSSD (28.7±0.2 g/day, n≥5) (
CD, conventional diet; HFSD, high-fat and high-sucrose diets; HFSSD, high-fat, high-sucrose and high-salt diets. Vertical dashed line indicates initial significant changes in SBP. Ten rats were used in each diet group for this statistical analysis across post diet days 0–120. *
SBP of the three groups were measured every 5 days from the beginning till the end of the experimental observation. Rats fed CD showed a slight increase in SBP from PDD 5 to 120 within normal range (
The FPG of rats among the three groups was measured every 5 days from PDD 0 to 30, and PDD120 before termination of the experiment. Rats fed CD showed no change in FPG from PDD 0 to 20 followed by a slight elevation from PDD 25 to 120 (
The fasting plasma glucose (FPG, A), the plasma insulin (B), the free fatty acids (FFA, C) and the homeostasis model assessment insulin resistance (Homa-IR) index (D) are shown. At least five rats were used in each diet group for this statistical analysis across post diet days 0–120. *
In parallel with FPG, plasma insulin and FFA were also measured every 5 days from PDD 0 to 30, and again on PDD 120. In comparison with the rats fed CD, the plasma level of insulin was not significantly changed before PDD 15 in rats fed HFSD and before PDD 20 in rats fed HFSSD (
The plasma levels of FFA were also unchanged until PDD 20 in rats fed both HFSD and HFSSD compared to the CD group (
HOMA-IR index was calculated based upon the concentrations of FPG and plasma insulin and this measure is thought to represent insulin sensitivity (
A parallel evaluation of somatic pain sensitivity was performed in each group of rats and bilateral PWMT and PWTL were measured over the whole time course. In rats fed CD, neither PWMT nor PWTL was significantly altered from PDD 0 to 120 (
The somatic sensory function was shown by changes in mechanical pain sensitivity (A-B) and thermal pain sensitivity (C-D) in bilateral hind paws (A and C for left hindpaw and B and D for right hindpaw). PWMT, paw withdrawal mechanical threshold; PWTL, paw withdrawal thermal latency. Five to six rats were used in each diet group for this statistical analysis across post diet days 0–120. *
To exclude motor modulation of sensory input, motor coordinating performance was measured on PDD 120 after sensory evaluation (
The somatic motor function was shown by motor coordinating performance of rats on a treadmill on post diet days 120. Five to six rats were used in each diet group for this statistical analysis. *
The ultrastructure of bilateral sciatic nerves of rats fed CD (n = 3), HFSD (n = 3) and HFSSD (n = 3) were examined after PDD 120 under EM. As shown in
Electron microscopic photomicrographs show the cross-section of the sciatic nerve fibers in the CD (A and D), HFSD (B and E) and HFSSD (C and F) groups. Dramatic pathological changes characterized by myelin breakdown or disruption and axon degeneration are mainly seen in large myelinated fibers (LMF) of rats fed HFSD (B) and HFSSD (C) when comparing with CD (A). The diets-induced LMF myelin changes are often seen as myelin lamina rarefaction, focal demyelination and vacuolization (yellow arrowheads in B-C). Axon degeneration of LMFs is characterized by abnormal high electron density and axonal plasmic shrinkage (asterisks in B-C). Ultrastructures of small myelinated fibers (SMF) and unmyelinated C fibers (UMF) in three groups are also shown (D-F). The axolemma and the Schwann cell covering are well maintained in all UMFs of rats fed CD (see red arrows in D), however, the axolemma and the Schwann cell membrane are thickened and perturbed shown as high electron density in both HFSD and HFSSD (red arrows in E-F). In addition, enlarged mitochondria and lipofuscin depositions are also seen in UMF axons of high energy/salt-treated rats (see yellow arrowheads in E-F) but not in control rats (D). The ultrastructures of SMFs in HFSD and HFSSD are well preserved when compared with CD (see yellow arrows in B-F), however, broken SMF can also be seen in the diet rats (double yellow arrows in E). My, myelin sheath; SC, the nucleus of Schwann cells; other abbreviations see
(A) Percent damage to LMF, SMF and UMF. (B) Distributional histograms of profile areas of myelinated fibers. (C) Boxplots show changes in profile ratios (changes in myelin profile) obtained by dividing area of axon profiles with area of fiber profiles of the myelinated fibers. (D) Proportion of nerve fibers with different pathologically-classified grades (grades pI-IV, for details see
However, compared with the severe disruptions present in the LMF, only mild pathological alterations were found in the SMF and the UMF of the sciatic nerves in rats fed HFSD and HFSSD compared to control (
Quantitative analysis of structural changes in myelinated fibers in the sciatic nerves of rats fed CD, HFSD and HFSSD are shown in
Under EM, the damaged fibers were classified into four grades according to the intensity and extensity of destruction of myelin sheath and axons within the sciatic nerve (
The spinal dorsal column is mainly comprised of central axonal fibers of the primary sensory neurons that innervate peripheral sensory organs perceiving cutaneous touch, pressure and vibration or deep proprioception. The ultrastructure of spinal dorsal column fibers in rats fed CD (n = 3), HFSD (n = 3) and HFSSD (n = 3) were examined as well after PDD 120 under EM. As shown in
Electron microscopic photomicrographs show the cross-section of the DC fibers in the CD (A), HFSD (B) and HFSSD (C) groups. Dramatic pathological changes characterized by myelin breakdown or disruption (yellow arrowheads), split between axon and myelin sheath (red arrowheads) and axon degeneration (asterisks) are mainly seen in large myelinated fibers of rats fed HFSD (B) and HFSSD (C) when comparing with CD (A). Beyond the pathological changes observed in the sciatic nerves (see legend in
As quantified in
(A) Percent damage to myelinated fibers. (B) Distributional histograms of profile areas of myelinated fibers. (C) Boxplots show changes in profile ratios of the myelinated fibers. (D) Proportion of nerve fibers with different pathologically-classified grades (grades pI-IV, for details see
Graded quantification of the amount of myelin damage in the spinal dorsal column (
During this longitudinal study of rats fed two kinds of high energy (HFSD and HFSSD), versus control diets
The timing of onset of each metabolic component induced by high fat/sugar or high fat/sugar/salt often differed with diet-type. Interestingly, the onset of hyperinsulinemia induced by the HFSD was on PDD 15, while the onset of the FFA rise occurred on PDD 20 in rats fed both the HFSD and HFSSD. The occurrence of hyperinsulinemia earlier than FFA dyslipidemia may contradict the existing hypothesis that dyslipidemia (with production of more FFA) may be the cause of IR
Here we show that the occurrence of painful polyneuropathy may reflect an early consequence of diet-induced metabolic syndrome (hypertension, hyperinsulinemia, and dyslipidemia) and prediabetes (IFG or IGT).
Relative to CD, both the HFSD and HFSSD could induce bilateral reduction in PWMT but with no accompanying changes in PWTL. These results suggest the occurrence of mechanical hypersensitivity (hyperalgesia and allodynia) but not thermal hypersensitivity in rats fed high energy/salt diets. However, rats fed HFSD showed greater (about 2 fold more) mechanical hypersensitivity than those fed HFSSD, suggesting that the high salt-induced hypertension, or other effects, might produce a protective anti-hyperalgesic phenotype on the HFSD-induced sensory dysfunction
Motor coordinating performance of rats with HFSD and HFSSD, measured by Rota Rod treadmill on the last day of the experiment (PPD 120) declined significantly when compared with rats fed CD. The impaired motor coordinating performance by high energy/salt diets could be attributable to damage to either peripheral motor fibers in the sciatic nerve, the somatic proprioceptive ascending projection fibers along the spinal dorsal column or the central motor system after chronic exposure to hyperinsulinemia, dyslipidemia (FFA), and IFG/or IGT
Painful peripheral neuropathy is a major type of neuropathic pain that has been recently redefined by NeuPSIG (Neuropathic Pain Special Interest Group of the International Association for the Study of Pain) as ‘pain arising as a direct consequence of a lesion or disease affecting the somatosensory system’
The primary finding of the current study is that high energy/salt diets can induce disruption or breakdown of myelin sheath and axonal degeneration in the LMF of both the peripheral and central branches of the dorsal root ganglion neurons. As damage to large-myelinated motor fibers in the sciatic nerves could not be excluded, motor neuropathy is also possible and indeed suggested by reduced motor activity of both high energy/salt diet animals relative to controls. In contrast, the SMF and the UMF in the sciatic nerves remained mostly intact in rats fed HFSD and HFSSD although axolemma thickening and intra-axonal plasmic lipofuscin depositions could be occasionally found in UMF, especially in rats fed HFSSD. Similarly, the damage to the LMF was more extensive in rats fed HFSSD than those fed HFSD, suggesting that besides high energy components of the diets, high dietary sodium and/or its resultant hypertension are probably other important factors contributing to the pathogenesis of the LMF neuropathies. Even though levels of LMF damage were highest in the HFSSD rats, the onset and level of peripheral neuropathy (measured as tactile but not thermal hypersensitivity) was lower in these animals, suggesting that a high-salt diet maybe in some way protective to levels of pain-like tactile allodynia experienced by the rats, but not to LMF damage. At first these results may seem contradictory, however patients with similar amounts of nerve damage can experience markedly different levels of chronic pain, and the mechanisms that create these disparities are poorly understood
Here we show pre-diabetic associated myelin damage in the dorsal columns of the spinal cord, however it has recently been recognized that IR may also lead to myelin injury or white matter lesions in the brain
In the current study, early metabolic syndrome (hyperinsulinemia, dyslipidemia, and hypertension) and prediabetic conditions (IFG) could be induced by high energy (high-fat and high-sucrose) diets in rats which later developed painful polyneuropathy that was characterized by myelin breakdown and LMF loss in both peripheral and central branches of primary afferent neurons. However, SMF and UMF were far less damaged in the same rats. The phenomenon that the high energy diets only induce mechanical, but not thermal, pain hypersensitivity may reflect a selective damage to LMF, but not to the SMF and UMF. Moreover, dietary sodium (high-salt) deteriorates the neuropathic pathological process induced by high energy diets further, but paradoxically high salt consumption may improve, at least temporarily, chronic pain perception in these animals.
We have therefore established a strong link between high-energy/high-salt diet induced metabolic syndrome and prediabetes which results in relatively selective LMF damage in both the PNS and CNS that in turn can result in neuropathic pain. These results have a profound impact on patient welfare relative to diet choice, not just for T2DM onset, but also for its associated neuropathic symptoms.
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The authors are grateful to Drs. Z Li, Y-Q Yu, W Sun for their kindly technical assistance, and to X-B Li, Y-J Yin and Y-Y An for facility supports, and to X-L Wang and Y Yang for SPF animal supplies, and to C-L Li, Z-Y Zhao, Y-F Lu, F Yang, Y Xu, J-H Jin, X-Y Cui for collaborations.