Apoptotic Stress Is Counterbalanced by Survival Elements Preventing Programmed Cell Death of Dorsal Root Ganglions in Subacute Type 1 Diabet
http://www.100md.com
《糖尿病学杂志》
1 Department of Pathology, Wayne State University School of Medicine, Detroit, Michigan
2 Morris Hood, Jr. Comprehensive Diabetes Center, Wayne State University School of Medicine, Detroit, Michigan
3 Department of Neurology, Wayne State University School of Medicine, Detroit, Michigan
CGRP, calcitonin geneeCrelated peptide; DPN, diabetic polyneuropathy; DRG, dorsal root ganglion; HSP, heat shock protein; NGF, nerve growth factor; SP, substance P; STZ-D, streptozotocin-induced diabetes
ABSTRACT
Several groups have reported apoptosis of dorsal root ganglion (DRG) cells as a prominent feature of diabetic polyneuropathy (DPN), although this has been controversial. Here, we examined subacute (4-month) type 1 diabetic BB/Wor rats with respect to sensory nerve functions, DRG and sural nerve morphometry, pro- and antiapoptotic proteins, and the expression of neurotrophic factors and their receptors. Sensory nerve conduction velocity was reduced by 13% and was accompanied by significant hyperalgesia. The numbers of DRG neurons including substance PeCand calcitonin geneeCrelated peptideeCpositive neurons were not altered, although they showed significant atrophy. Sural nerve morphometry showed decreased numbers of myelinated and unmyelinated fibers. Active caspase-3 and Bax expressions were increased, whereas antiapoptotic Bcl-xl and heat shock protein (HSP) 27 expressions in DRGs were increased. Nerve growth factor (NGF) contents in sciatic nerves and the expression of NGF receptor TrkA in DRGs were decreased. Immunohistochemistry showed increased numbers of active caspase-3eC, HSP70-, and HSP27-positive neurons. Examinations of DRGs revealed no structural evidence of apoptosis but rather progressive hydropic degenerative changes. We conclude that apoptotic stress is induced in DRGs but is counterbalanced by survival elements in subacute type 1 diabetic BB/Wor rats and that distal nerve fiber loss reflects a dying-back phenomenon caused by impaired neurotrophic support.
Diabetic polyneuropathy (DPN) is the most common late complication of diabetes and shows an increasing prevalence. Like other diabetes complications, DPN has been ascribed to hyperglycemia and subsequent metabolic abnormalities, particularly oxidative stress. Although other factors contribute to its development, such as insulin and C-peptide deficiencies and consequent deprivation of neurotrophic support (1), these have received less attention. Several investigators have reported neuronal apoptosis in relatively acute (2- to 3-month) streptozotocin-induced diabetes (STZ-D) in rats, sometimes exceeding 30% of dorsal root ganglion (DRG) cells as a contributing factor to DPN (2eC4), while others have reported no significant neuronal loss in DRGs even after 12 months of STZ-D (5,6). The validity of major programmed cell death can be disputed, since the magnitude of reported apoptotic neuronal death does not correlate with a corresponding loss of axonal extensions in the peripheral nerve of the STZ-D rat. In contrast to the STZ-D rat, the spontaneously type 1 diabetic BB/Wor rat develops progressive sensory nerve fiber loss (7), as in humans, which therefore could reflect loss of parent DRG cells.
Apoptotic phenomena occur in pancreatic -cells in type 1 diabetes (8), in diabetic retinopathy (9), and in hippocampal neurons in primary diabetic encephalopathy (10,11). Programmed cell death is believed to result from hyperglycemia-induced mitochondrial dysfunction (4,12) and depletion of antiapoptotic trophic factors like IGFs, insulin, and C-peptide and downregulation of their receptors (13,14). Apoptogenic stresses are mediated via increased expression of proapoptotic proteins such as Bax, oxidative DNA damage, activation of death receptors, and various upstream caspases inducing activation of caspase-3 as well as noncaspase-dependent apoptotic stressors (11). Proapoptotic activities are counteracted by stress-induced intrinsic antiapoptotic proteins, such as mitochondrial Bcl-xl, the DNA repair enzyme poly (ADP-ribose) polymerase (PARP) (5), and heat shock proteins (HSPs) (15,16).
Here, we examined subacute (4-month) spontaneously diabetic BB/Wor rats, a close model of human type 1 diabetes (17), with respect to sensory nerve functions, DRG and sural nerve morphometry and morphology, expression of active caspase-3, pro- and antiapoptotic proteins, HSPs, and trophic factors and their receptors in DRGs. Particular attention was paid to nociceptive DRG ganglion cells and their content of substance P (SP) and calcitonin geneeCrelated peptide (CGRP), since these appear to be particularly vulnerable under diabetic conditions (18,19).
RESEARCH DESIGN AND METHODS
Pre-diabetic BB/Wor rats and age-matched nondiabetes-prone BB rats were obtained from Biomedical Research Models, Worcester, Massachusetts. All rats had free access to water and rat diet. Body weight, urine volume, and glucosuria (Keto-Diastix; Bayer, Elkhart, IN) were monitored daily to ascertain onset of diabetes and for titration of daily insulin doses. After onset of diabetes, diabetic rats received titrated doses (0.5eC3.0 units/day) of protamine zinc insulin (Novo Nordisk, Princeton, NJ) to maintain blood glucose levels at 25 mmol/l glucose and to prevent ketoacidosis. Blood glucose levels were measured biweekly. Animals were cared for in accordance with guidelines of the Animal Investigation Committee, Wayne State University, and those of the National Institutes of Health (publ. no. 85-23, 1995).
Measurement of plasma insulin level.
Insulin levels were measured in plasma obtained at the time the rats were killed, 20 h after the last insulin injection, using an enzyme-linked immunosorbent assay kit (Linco Research, St. Charles, MO).
Electrophysiological recordings.
Rats were anesthetized with intraperitoneal pentobarbital sodium (50 mg/kg body wt). Rectal temperature was maintained at 36eC37°C by a heating pad and monitored by a rectal probe. Sensory nerve conduction velocity was recorded in the left hind limb as previously described (18).
Thermal plantar test.
Latencies of hind-paw withdrawal to thermal stimulation (42°C; 152 mW/cm2) was measured monthly (UGO Biological Research, Comerio, Italy) (18). The time from heat source activation to the animal’s self-withdrawal in seconds was measured six times in alternating hind paws. The mean of these measurements was calculated and used as the measure of the withdrawal latency.
Tissue collection.
Four months after onset of diabetes, animals were anesthetized. Three control and BB/Wor rats were perfused with 500 ml of 4% paraformaldehyde fixative. The left L5 DRGs were postfixed in situ for 10 min with 1% cacodylate-buffered (pH 7.4) 2.5% glutaraldehyde then excised and immersed in the same fixative for 2 h at 4°C. They were postfixed in 1% cacodylate-buffered (pH 7.4) osmium tetroxide, dehydrated, and embedded in Epon for morphologic and morphometric analyses. DRGs of the right side were fixed by immersion in 4% paraformaldehyde in 0.1 mol/l phosphate buffer (pH 7.4) overnight at 4°C, rinsed in PBS, dehydrated, immersed in xylene, and embedded in paraffin.
The right sural nerve was dissected from five diabetic and five control rats and fixed in 1% cacodylate-buffered (pH 7.4) 2.5% glutaraldehyde, dehydrated, and embedded in Epon for morphometric analysis as previously described (20,21). DRGs and sciatic nerves were collected from five diabetic and five control rats for protein extraction. Tissues were snap frozen in liquid nitrogen and kept in eC80°C until use.
Morphometry.
Morphometric analyses of DRG neurons were performed on toluidine-blueeCstained step sections (0.5 e蘭 thick; 40 e蘭 apart) using a video image analysis system (Image-Pro Plus 3.0; Media Cybernetics, Silver Spring, MD). L5 ganglia were sectioned, 35eC39 sections per ganglia. Neurons with distinguishable nuclei were counted and their area measured. Diameters of neuronal nuclei were measured and averaged. Number of neurons per DRG was calculated as the sum of numbers of neurons per section x 40/mean nuclear diameter.
Semithin (0.5-e蘭) cross-sections of Epon-embedded sural nerves were stained with toluidine-blue for light microscopic morphometric analysis using a computerized image analysis system (Image-1; Universal Imaging, West Chester, PA). This system is programmed to assess the total complement of sural nerve myelinated fibers and provides the following parameters: fasicular area (e蘭2), number of fibers (#), fiber density (#/mm2), mean fiber area (e蘭2), mean axonal area (e蘭2), mean myelin area (e蘭2), axon-to-myelin ratio, index of circularity, and fiber occupancy rate (in percent) as previously described (20,21).
For morphometric analyses of unmyelinated fibers, five animals per group were examined. Ultrathin cross-sections of sural nerves were obtained with the aid of an LKB ultramicrotome (Marviac Limited, Halifax, Canada) and stained with uranyl acetate and lead citrate. They were examined in a Zeiss EM 900 transmission electron microscope (Carl Zeiss, Oberkochen, Germany). Systematically selected frames representing 40eC50% of the sural nerve cross-sectional area were obtained. Photographs were enlarged 10,000 times, scanned, and downloaded to the computerized image analysis system. The following morphometric parameters of unmyelinated fibers were obtained: unmyelinated fiber number, fiber density (#/mm2), mean fiber size (e蘭2), axon numbers per Schwann cell unit and the frequencies of collagen pockets, denervated Schwann cell profiles, type 2 axon-Schwann cell relationship, and regenerating C-fibers (19,21).
Morphological changes.
Semithin (0.5-e蘭) toluidine-blueeCstained sections of DRGs were examined for margination of nuclear chromatin, apoptotic bodies, other degenerative changes, and nodules of Nageotte as an indication of neuronal loss.
Immunohistochemistry for SP, CGRP, HSP27, HSP70, and active caspase-3 in lumbar DRG.
Six-micrometer-thick sections were immunostained using an avidin biotin complex kit (Vector Laboratories, Burlingame, CA). Antibodies used are listed in Table 1. Quantifications of positive neurons were performed using the same image analysis system as above. Images of three serially sectioned DRG (60 e蘭 apart) were captured and assessed using a binary scale. In each ganglion, 200eC300 ganglion cells with visible nuclei were captured. The number and areas of SP- and CGRP-positive neurons were determined in each section. SP- and CGRP-positive neurons per ganglion were calculated as neurons per ganglion times positive neurons per section divided by total neurons per section. The frequencies of HSP27- and HSP70-positive neurons and caspase-3eCpositive neurons and neuronal nuclei were calculated from the same serial sections.
Measurement of SP and CGRP contents of DRGs.
Enzyme immunometric assays were used to assess SP (Cayman Chemical, Ann Arbor, MI) and CGRP (SPIbio, Massy Cedex, France) in DRGs. Results were expressed as picograms per ganglion (18).
Western blotting.
DRGs were lysed in detergent lysis buffer (50 mmol/l Tris-HCl, pH 7.4, 150 mmol/l NaC1, 1 mmol/l EDTA, 1% Triton X-100, 1 mmol/l phenylmethylsulfonyl fluoride, 1 e蘥/ml leupeptin, and 1 e蘥/ml aprotinin). The lysates were centrifuged at 14,000 rpm for 20 min at 4°C, and protein concentrations were measured using bicinchoninic acid protein assay reagent (Pierce, Rockford, IL) with BSA as standard. Ten to 40 e蘥 was separated by 7.5eC15% SDS-PAGE and transferred to polyvinylidine fluoride membranes (Bio-Rad, Hercules, CA). Membranes were blocked with Tween-20-TriseCbuffered saline (10-mmol/l Tris-HCl, pH 7.5, 100 mmol/l NaC1, and 0.1% Tween 20) containing 5% nonfat dry milk (Bio-Rad) before incubation with primary antibodies (Table 1). Antigen detection was performed using chemiluminescence (Amersham Pharmacia Biotech, Piscataway, NJ) with horseradish peroxidaseeCconjugated secondary antibodies. Membranes were exposed to Biomax film (Kodak, Rochester, NY). Images were scanned and densities determined by the Bio-Rad Fluoro-S multiimager (Bio-Rad). Expression of proteins was corrected for by actin density, and expression in control animals was arbitrarily set to 1.0.
Nerve growth factor in sciatic nerve.
Nerve growth factor (NGF) content was determined with a quantitative two-site enzyme immunoassay (Emax Immunoassay System; Promega, Madison, WI). Assays were performed according to manufacturers’ protocol. Preweighed (50 mg) sciatic nerve segments were homogenized in lysis buffer (137 mmol/l NaC1, 20 mmol/l Tris HCl, pH 8.0, 1% NP40, 10% glycerol, 1 mmol/l phenylmethylsulfonyl fluoride, 10 e蘥/ml aprotinin, 1 e蘥/ml leupeptin, and 0.5 mmol/l sodium vanadate) and centrifuged (14,000 rpm for 20 min). The supernatants were loaded on MaxiSorp 96-well plates (Nalge Nunc International, Rochester, NY), and measurements were performed as previously described (18,19). Results were expressed as picograms per milligram protein.
Statistical analysis.
All values are expressed as means ± SD. Significance of differences was analyzed by ANOVA. Group differences were assessed by Scheffes test. Significance was defined as a P value <0.05. All analyses were performed by personnel unaware of the animal identities.
RESULTS
Clinical findings.
At 4 months, BB/Wor rats showed severe hyperglycemia (control rats [n = 8]: 5.0 ± 0.4 mmol/l, diabetic rats [n = 8]: 24.4 ± 1.9 mmol/l; P < 0.001) and significant reductions in body weight (control rats [n = 8]: 458.1 ± 16.2 g, diabetic rats [n = 8]: 373.8 ± 16.4 g; P < 0.001). Serum insulin level was decreased in diabetic rats (control rats [n = 8]: 2.39 ± 0.28 ng/ml, diabetic rats [n = 8]: 0.67 ± 0.26 ng/ml; P < 0.001).
Functional data.
Sensory nerve conduction velocity decreased progressively from 91% (control rats [n = 8]: 39.8 ± 0.9 m/s, diabetic rats [n = 8]: 36.2 ± 1.2 m/s; P < 0.001) of normal at 2 months to 87% at 4 months (control rats [n = 8]: 40.9 ± 1.5 m/s, diabetic rats [n = 8]: 35.6 ± 1.6 m/s; P < 0.001). Hyperalgesia, reflecting unmyelinated fiber function, was measured as latencies of hind-paw withdrawal to thermal stimuli. Diabetic rats showed progressive and significant decreases in the latencies to thermal stimuli that reached 11.9 ± 2.7 s (n = 8) at 4 months compared with 22.7 ± 2.2 s in control rats (n = 8; P < 0.001).
Morphometry of DRG.
Neuronal size distribution of DRGs showed a shift toward smaller sizes in diabetic rats (Fig. 1A), consistent with a reduced mean neuronal size in diabetic rats (P < 0.05; Table 2). Although there was 14.7% fewer DRG neurons in diabetic rats, this did not reach statistical significance (Table 2). SP- and CGRP-positive neurons showed small shifts toward smaller sizes (Figs. 1B and C); however, the number of SP and CGRP neurons were not significantly altered (Table 2) in diabetic rats. Mean SP neuronal size was not significantly changed, whereas mean CGRP neuronal size was decreased by 15.6% (P < 0.05) in diabetic rats (Table 2).
Morphological changes.
Approximately 1,000 DRG neurons per ganglion were examined. The vast majority of ganglion cells showed normal morphology (Fig. 2A). No chromatin margination or apoptotic bodies were observed in diabetic or control rats. Instead, subplasmolemmal small vacuoles were observed in 10.5 ± 2.1 and 10.9 ± 1.7% of neurons (Fig. 2B) in control and diabetic rats, respectively. These coalesced to form larger cytoplasmic vacuoles in 0.31 ± 0.13% of neurons in diabetic rats (Fig. 2C) compared with 0.03 ± 0.06% (P < 0.05) in control rats. Nodules of Nageotte (Fig. 2D) were equally common in control and diabetic rats (0.17 ± 0.06 vs. 0.19 ± 0.13%). The structural abnormalities are consistent with hydropic degeneration.
Sural nerve morphometry.
The results of myelinated and unmyelinated fiber morphometry of sural nerves are given in Table 3. Myelinated fibers in diabetic rats showed a 15.6% fiber loss (P < 0.01) and decreased mean axonal (P < 0.05) and fiber (P < 0.005) size, whereas mean myelinated area was unchanged. The axonal atrophy was reflected by decreased index of circularity (P < 0.05).
Unmyelinated fiber morphometry of diabetic rats revealed a 28.5% fiber loss (P < 0.001), decreased fiber density (P < 0.05), axonal atrophy (P < 0.05), and decreased number of axons per Schwann cell unit (P < 0.05). These changes were accompanied by increased frequencies of denervated Schwann cell profiles (P < 0.005), collagen pockets (P < 0.05), type 2 Schwann cell/axon relationships (P < 0.001), and regenerating fibers (P < 0.001) in diabetic rats.
Expression of active caspase-3 and caspase 3eCpositive neurons.
Western blots for active caspase-3 showed increased (P < 0.05) levels in diabetic DRGs (Fig. 3A). Immunohistochemically, the frequencies of caspase-3eCpositive DRG neurons showing cytoplasmic staining (Fig. 4A) and nuclear staining (Fig. 4B) were increased (P < 0.001 and P < 0.05, respectively) in diabetic rats.
Expression of pro- and antiapoptotic elements.
Proapoptotic Bax was increased 1.9-fold (P < 0.005) in diabetic rats (Fig. 3B). This was associated with a 1.6-fold (P < 0.05) increase in antiapoptotic Bcl-xl in diabetic ganglion cells (Fig. 3C). The expression of the low-affinity NGF receptor (NGF p75R), Fas, PARP, cleaved PARP, or caspase-12, reflective of endoplasmic reticulum dysfunction, were not altered (Figs. 3DeCG).
Neurotrophic factors.
The NGF content in the sciatic nerve was significantly decreased (P < 0.05) in diabetic rats (Fig. 5H). The high-affinity NGF receptor (TrkA) expression in diabetic DRGs was significantly decreased (P < 0.05; Fig. 5E), whereas the IGF-1 receptor and insulin receptor expressions in DRGs were not altered (Figs. 5F and G).
SP and CGRP contents in DRGs.
SP and CGRP contents in DRGs were not significantly changed in diabetic rats (Table 2).
HSPs in DRGs.
HSP27 protein expression was significantly (P < 0.05) increased in diabetic DRGs (Fig. 5A). This change correlated with an increased frequency (P < 0.05) of immunohistochemically positive neurons (Fig. 6A). HSP27-positive staining was observed in the cytoplasm of neurons and axons of all sizes. The expression of inducible HSP70 was not altered in diabetic DRGs (Fig. 5B). Immunostaining of HSP70 in DRGs showed localization mainly to Schwann cells, satellite cells, and small neurons (Fig. 6B). Although the percentage of HSP70-positive neurons was low, it was significantly increased (P < 0.05) in diabetic DRGs. Constitutively expressed HSC70 or HSP40 were not altered (Figs. 5C and D).
DISCUSSION
Human DPN shows progressive loss of myelinated and unmyelinated sensory fibers in peripheral nerves, accompanied by degeneration and loss of parent DRG neurons, and is classified as a peripheral axonopathy of dying-back type (1,7,22eC24). Hyperglycemia with consequent perturbations of the polyol pathway, nerve hypoxemia, and oxidative stress have been suggested as the main culprits (1). In type 1 DPN, impaired insulin and C-peptide signaling plays contributing roles, causing perturbed gene regulation of neurotrophic factors and their receptors and aberrations of the expression and posttranslational modifications of neuroskeletal and adhesive proteins (1,18,19,24,25). It has been claimed that apoptosis of sensory ganglion cells is a prominent component in the pathogenesis of DPN in STZ-D rats (2eC4,12).
Using the spontaneously type 1 diabetic BB/Wor rat we demonstrate here 1) increased stainability and upregulation of active caspase-3 and proapoptotic Bax in diabetic DRGs, 2) that these apoptotic stresses are accompanied by the induction of antiapoptotic Bcl-xl and survival promoting HSP27 and HSP70, 3) that NGF content in sciatic nerve and high-affinity NGF receptor TrkA expression in diabetic DRGs are decreased, 4) that morphometric analyses of sural nerve reveal myelinated and unmyelinated fiber loss and axonal atrophy in diabetic rats, and 5) that morphometric and morphologic examinations of DRGs show no neuronal loss or qualitative changes indicative of apoptosis. Instead, DRG neurons show progressive hydropic damage similar to that described in human DPN (22).
Caspase-3 is the common final executing caspase and is activated by several upstream apoptotic pathways. Here, we demonstrate increased expression of active caspase-3 as well as increased stainability in neuronal cytoplasm and nuclei in diabetic rats. These findings are in agreement with previous data (2,5,6) and were accompanied by increased expression of Bax, indicative of mitochondrial dysfunction. On the other hand, these apoptotic stressors were associated with upregulation of antiapoptotic Bcl-xl and HSP27 as well as an increased number of neurons staining positive for HSP70.
HSPs are proteins induced by thermal stimuli and act as chaperones to support folding of proteins. HSP27 and HSP70 have been widely investigated, and their protective effect against neuronal death has been repeatedly reported (15,16,26eC30). HSP27 is constitutively expressed and can be induced not only by thermal stimuli but also by trophic factor withdrawal or by apoptotic stress (26). HSP27 interacts with cytochrome c, blocking its interaction with Apaf-1, procaspase 9, and downstream activation of caspase-3 (26), and it inhibits Fas-induced caspase-independent apoptosis by interacting with Daxx (30). However, Fas was not increased in DRGs in this study, suggesting that caspase-independent mechanisms are not involved. Inducible HSP70 is a survival element (28,29) induced by several stresses (31). In this study, we found localization of HSP70 in Schwann cells and small neurons. Although its expression in DRGs was not changed, immunohistochemistry revealed increased HSP70 positivity in small DRG neurons. HSP70 may therefore be increased in the more susceptible small nociceptive neurons under diabetic conditions. In keeping with the present findings, Guzhova et al. (32) reported that HSP70 may be exported from glia cells and that extracellular HSP70 has protective effects on neurons.
Taken together with the morphometric and morphologic data, which failed to demonstrate significant neuronal loss and qualitative changes suggestive of ongoing apoptosis, it appears that apoptotic stresses are induced in DRGs but that the final execution of DRG neuronal apoptosis is prevented by the induction of counterregulatory elements such as Bcl-xl and HSPs. This is supported by the findings that the common death substrate protein PARP and its cleavage product, indicative of DNA damage, were unaltered in diabetic DRGs.
These findings differ from those previously reported in the hippocampus of the same rat model (11), in which activation of caspase-3 and increased expression of Bax were not counteracted by induction of Bcl-xl. Futhermore, activation of FAS and the death-receptor NGF-p75R as well as increased expression of cleaved PARP reported in hippocampus could not be demonstrated in DRGs. The apoptotic phenomena seen in hippocampus were associated with significant neuronal loss (11), which therefore suggest that diabetes induces a more severe apoptotic stress in central neuronal populations than in peripheral DRG neurons.
Instead, DRG neurons showed atrophy and vacuolar cytoplasmic degeneration, characteristic of hydropic degeneration. The frequency of nodules of Nageotte, signifying ganglion cell loss, was not increased in diabetic rats. The mechanisms underlying these changes are not known. However, it is likely that impaired synthesis of neuroskeletal proteins previously demonstrated in this model (33) may account for not only axonal atrophy but also for the atrophy of neuronal perikaria as demonstrated here (24). The vacuolar changes are likely to reflect impaired neuronal energy metabolism and perturbed sodium handling possibly via impaired Na+/K+-ATPase activity and/or decreased DRG blood flow (34) and may therefore be analogous to nodal axonal swellings seen in peripheral nerve fibers in DPN (35). These constructs are consistent with impaired insulin/C-peptide action affecting Na+/K+-ATPase activity and resulting in downregulation of NGF and suppression of its high-affinity TrkA receptor as demonstrated here (18,19,36eC38).
Abnormalities in insulin and NGF signaling but not in that of IGF-1 may account for the greater susceptibility of C-fibers and their parent NGF-dependent neurons in DRGs. This is supported by the findings in the isohyperglycemic and hyperinsulinemic type 2 counterpart to this type 1 diabetes model, in which perturbations of NGF and its high-affinity receptor are milder and associated with less severe C-fiber pathology (19).
Increased hyperalgesia, as reported here and previously (18,19,21), has been associated with increased spontaneous firing of nociceptive neurons (39) due to upregulation of 3 and tetrodotoxin-resistant voltage-gated Na+ channels in small nociceptive and A DRG neurons (40,41). This will inevitably result in increased Na+ influx as demonstrated by increased Na+ current densities and a shift in steady-state inactivation in diabetic dorsal root ganglia (41). Coupled with perturbed Na+/K+-ATPase activity, these changes may not only account for the hyperexcitability of nociceptive DRGs but also for the observed progressive hydropic degenerative changes. Thermal hypoalgesia is observed in humans and STZ-D rats with DPN. In STZ-D rats, this is proceeded by a short period of hyperalgesia (42eC45) analogous to a transient period of hyperalgesia in BB/Wor rats (18,19). The transient hyperalgesia most likely corresponds to anatomically intact hyperexitable C-fibers, whereas the ensuing hypoalgesia probably reflects the progressive denervation by C-fibers due to the dying-back process.
In summary, in the present study we could not confirm previous reports on high apoptotic activity and cell death in DRGs. Instead, the present data are consistent with those reported by Cheng and Zochodne (5), who failed to show loss of DRG neurons in 12-month STZ-D rats. The data are also in keeping with those reported by Burnand et al. (46), who failed to demonstrate the induction of apoptosis-related genes in acutely (8 weeks) STZ-D rats. The axonal loss and DRG neuronal atrophy reported here are consistent with an axonal dying-back phenomenon due to impaired cytoskeletal protein synthesis (6,7,33) secondary to insulin/C-peptide deficiencies and deprived neurotrophic support.
ACKNOWLEDGMENTS
This study was supported by research grants from the Thomas Foundation, which is also providing support to the postdoctoral fellowship of H.K., and the Juvenile Diabetes Research Foundation.
REFERENCES
Sima AAF: New insights into the metabolic and molecular basis for diabetic neuropathy. Cell Mol Life Sci60 :2445 eC2464,2003
Schmeichel AM, Schmetzer JD, Low PA: Oxidative injury and apoptosis of dorsal root ganglion neurons in chronic experimental diabetic neuropathy. Diabetes52 :165 eC171,2003
Russel JW, Sullivan KA, Windebank AJ, Herrmann DN, Feldman EL: Neurons undergo apoptosis in animal and cell culture models of diabetes. Neurobiol Dis6 :347 eC363,1999
Scrinivasan S, Stevens M, Wiley JW: Diabetic peripheral neuropathy: evidence for apoptosis and associated mitochondrial dysfunction. Diabetes49 :1932 eC1938,2000
Cheng C, Zochodne DW: Sensory neurons with activated caspase-3 survive long-term experimental diabetes. Diabetes52 :2363 eC2371,2003
Zochodne DW, Verge VM, Cheng C, Sun H, Johnston J: Does diabetes target ganglion neurones Progressive sensory neurone involvement in long-term experimental diabetes. Brain124 :2319 eC2334,2001
S ima AA, Bouchier M, Christensen H: Axonal atrophy in sensory nerves of the diabetic BB-Wistar rat: a possible early correlate of human diabetic neuropathy. Ann Neurol13 :264 eC272,1983
Thompson CB: Apoptosis in the pathogenesis and treatment of disease. Science267 :1456 eC1462,1995
Barber AJ, Lieth E, Khin SA, Antonetti DA, Buchanan AG, Gardner TW: Neural apoptosis in the retina during experimental and human diabetes: early onset and effect of insulin. J Clin Invest102 :783 eC791,1998
Li ZG, Zhang W, Grunberger G, Sima AA: Hippocampal neuronal apoptosis in type 1 diabetes. Brain Res946 :221 eC231,2002
Sima AAF, Li Z-G: The effect of C-peptide on cognitive dysfunction and hippocampal apoptosis in type 1 diabetes. Diabetes54 :1497 eC1505,2005
Vincent AM, Brownlee M, Russel JW: Oxidative stress and programmed cell death in diabetic neuropathy. Ann N Y Acad Sci959 :368 eC383,2002
Russel JW, Feldman EL: Insulin-like growth factor-1 prevents apoptosis in sympathetic neurons exposed to high glucose. Horm Metab Res31 :90 eC96,1999
Li Z-G, Zhang W, Sima AAF: C-peptide enhances insulin-mediated cell growth and protection against high glucose induced apoptosis in SH-SY5Y cells. Diabetes Metab Res Rev19 :375 eC385,2003
Li C-Y, Lee J-S, Ko Y-G, Kim J-I, Seo J-S: Heat shock protein 70 inhibits apoptosis downstream of cytochrome c release and upstream of caspase-3 activation. J Biol Chem275 :25665 eC25671,2000
Lewis SE, Mannion RJ, White FA, Coggeshall RE, Beggs S, Costigan M, Martin JL, Dillmann WH, Woolf CJ: A role for HSP27 in sensory neuron survival. J Neurosci19 :8945 eC8953,1999
Sima AAF: Diabetes underlies common neurological disorders (Editorial). Ann Neurol56 :459 eC461,2004
Kamiya H, Zhang W, Sima AAF: C-peptide prevents nociceptive sensory neuropathy in type 1 diabetes. Ann Neurol56 :827 eC835,2004
Kamiya H, Murakawa Y, Zhang W, Sima AAF: Unmyelinated fiber sensory neuropathy differs in type 1 and type 2 diabetes. Diabetes Metab Res Rev21 :448 eC458,2005
Sima AAF, Zhang W, Sugimoto K, Henry D, Li Z, Wahren J, Grunberger G: C-peptide prevents and improves chronic type 1 diabetic neuropathy in the BB/Wor-rat. Diabetologia44 :889 eC897,2001
Murakawa Y, Zhang W, Pierson CR, Brismar T, Ostenson CG, Efendic S, Sima AAF: Impaired glucose tolerance and insulinopenia in the GK-rat causes peripheral neuropathy. Diabetes Metab Res Rev18 :473 eC483,2002
Greenbaum D, Richardson PC, Salmon MW, Urich, H: Pathologic aberrations on six cases of diabetic neuropathy. Brain87 :201 eC214,1964
Sima AAF, Nathaniel V, Bril V, McEwen TAJ, Greene DA: Histopathological heterogeneity of neuropathy in insulin-dependent and noninsulin-dependent diabetes, and demonstration of axo-glial dysjunction in human diabetic neuropathy. J Clin Invest81 :349 eC364,1988
Scott JN, Clark AW, Zochodne DW: Neurofilament and tubulin gene expression in progressive experimental diabetes: failure of synthesis and export by neurons. Brain122 :2109 eC2117,1999
Sima AAF, Zhang W, Li Z-G, Murakawa Y, Pierson CR: Molecular alterations underlie nodal and paranodal degeneration in type 1 diabetic neuropathy and are prevented by C-peptide. Diabetes53 :1556 eC1563,2004
Benn SC, Perrelet D, Kato AC, Scholz J, Decosterd I, Mannion RJ, Bakowska JC, Woolf CJ: Hsp27 upregulation and phosphorylation is required for injured sensory and motor neuron survival. Neuron36 :45 eC56,2002
Gurbuxani S, Schmitt E, Cande C, Parcellier A, Hammann A, Daugas E, Kouranti I, Spahr C, Pance A, Kroemer G, Garrido C: Heat shock protein 70 binding inhibits the nuclear import of apoptosis-inducing factor. Oncogene22 :6669 eC6678,2003
Beere HM, Wolf BB, Cain K, Mosser DD, Mahboubi A, Kuwana T, Tailor P, Morimoto RI, Cohen GM, Green DR: Heat shock protein 70 inhibits apoptosis by preventing recruitment of procaspase-9 to the Apaf-1 apoptosome. Nat Cell Biol2 :469 eC475,2000
Ravagnan L, Gurbuxani S, Susin SA, Maisse C, Daugas E, Zamzami N, Mak T, Jaattela M, Penninger JM, Garrido C, Kroemer G: Heat shock protein 70 antagonizes apoptosis-inducing factor. Nat Cell Biol3 :839 eC843,2001
Mehlen P, Schulzeosthoff K, Arrigo AP: Small stress proteins as novel regulators of apoptosis: heat shock protein 27 blocks Fas/APO-1 and stanrosporine-induced cell death. J Biol Chem271 :16510 eC16514,1996
Yenari MA: Heat shock proteins and neuroprotection. Adv Exp Med Biol513 :281 eC299,2002
Guzhova I, Kislyakova K, Moskaliova O, Fridlanskaya I, Tytell M, Cheetham M, Margulis B: In vitro studies show that Hsp70 can be released by glia and that exogenous Hsp70 can enhance neuronal stress tolerance. Brain Res914 :66 eC73,2001
Xu G, Pierson CR, Murakawa Y, Sima AAF: Altered tubulin and neurofilament expression and impaired axonal growth in diabetic nerve regeneration. J Neuropath Exp Neurol61 :164 eC175,2002
Zochodne DW, Ho LT, Allison JA: Dorsal root ganglia microenvironment of female BB Wistar diabetic rats with mild neuropathy. J Neurol Sci127 :36 eC42,1994
Sima AAF, Brismar T: Reversible diabetic nerve dysfunction: structural correlates to electrophysiological abnormalities. Ann Neurol18 :21 eC29,1985
Reico-Pinto E, Lang FF, Ishii DN: Insulin and insulin-like growth factor II permit nerve growth factor binding and the neurite formation response in cultured neuroblastoma cells. Proc Natl Acad Sci U S A81 :2562 eC2566,1984
Fernyhough P, Diemel LT, Brewster WJ, Tomlinson DR: Deficits in sciatic nerve neuropeptide content coincide with a reduction in target tissue nerve growth factor messenger RNA in streptozotocin-diabetic rats: effects of insulin treatment. Neurosci62 :337 eC344,1994
Zhang W, Yorek M, Pierson CR, Murakawa Y, Breidenbach A, Sima AA: Human C-peptide dose dependently prevents early neuropathy in the BB/Wor-rat. Int J Exp Diabetes Res2 :187 eC193,2001
Burchiel KJ, Russell LC, Lee RP, Sima AAF: Spontaneous activity of primary afferent neurons in diabetic BB/Wistar rats. Diabetes34 :1210 eC1213,1985
Shah BS, Gonzalez I, Bramwell S, Pinnock RD, Lee K, Dixon AK: 3, a novel auxillary subunit for the voltage gated sodium channel is upregulated in sensory neurons following streptozotocin induced diabetic neuropathy in rat. Neurosci Lett309 :1 eC4,2001
Hirade M, Yasuda H, Omatsu-Kanbe M, Kikkawa R, Kitasato H: Tetrodotoxin-resistant sodium channels of dorsal root ganglion neurons are readily activated in diabetic rats. Neurosci90 :933 eC939,1999
Li F, Obrosova IG, Abatan O, Tian D, Larkin D, Stuenkel EL, Stevens MJ: Taurine replacement attenuates hyperalgesia and abnormal calcium signaling in sensory neurons of STZ-D rats. Am J Physiol Endocrinol Metab288 :E29 eCE36,2005
Cameron NE, Tuck Z, McCabe L, Cotter MA: Effect of the hydroxyl radical scavenger, dimethylthiourea, on peripheral nerve tissue perfusion, conduction velocity and nociception in experimental diabetes. Diabetologia44 :1161 eC1169,2001
Fox A, Eastwood C, Gentry C, Manning D, Urban L: Critical evaluation of the streptozotocin model of painful diabetic neuropathy in the rat. Pain81 :307 eC316,1999
Calcutt NA, Freshwater JD, Mizisin AP: Prevention of sensory disorders in diabetic Sprague-Dawley rats by aldose reductase inhibition or treatment with ciliary neurotrophic factor. Diabetologia47 :718 eC724,2004
Burnand RC, Price SA, McElhaney M, Barker D, Tomlinson DR: Expression of axotomy-inducible and apoptosis-related genes in sensory nerves of rats with experimental diabetes. Brain Res Mol Brain Res20 :235 eC240,2004(Hideki Kamiya, Weixian Zh)
2 Morris Hood, Jr. Comprehensive Diabetes Center, Wayne State University School of Medicine, Detroit, Michigan
3 Department of Neurology, Wayne State University School of Medicine, Detroit, Michigan
CGRP, calcitonin geneeCrelated peptide; DPN, diabetic polyneuropathy; DRG, dorsal root ganglion; HSP, heat shock protein; NGF, nerve growth factor; SP, substance P; STZ-D, streptozotocin-induced diabetes
ABSTRACT
Several groups have reported apoptosis of dorsal root ganglion (DRG) cells as a prominent feature of diabetic polyneuropathy (DPN), although this has been controversial. Here, we examined subacute (4-month) type 1 diabetic BB/Wor rats with respect to sensory nerve functions, DRG and sural nerve morphometry, pro- and antiapoptotic proteins, and the expression of neurotrophic factors and their receptors. Sensory nerve conduction velocity was reduced by 13% and was accompanied by significant hyperalgesia. The numbers of DRG neurons including substance PeCand calcitonin geneeCrelated peptideeCpositive neurons were not altered, although they showed significant atrophy. Sural nerve morphometry showed decreased numbers of myelinated and unmyelinated fibers. Active caspase-3 and Bax expressions were increased, whereas antiapoptotic Bcl-xl and heat shock protein (HSP) 27 expressions in DRGs were increased. Nerve growth factor (NGF) contents in sciatic nerves and the expression of NGF receptor TrkA in DRGs were decreased. Immunohistochemistry showed increased numbers of active caspase-3eC, HSP70-, and HSP27-positive neurons. Examinations of DRGs revealed no structural evidence of apoptosis but rather progressive hydropic degenerative changes. We conclude that apoptotic stress is induced in DRGs but is counterbalanced by survival elements in subacute type 1 diabetic BB/Wor rats and that distal nerve fiber loss reflects a dying-back phenomenon caused by impaired neurotrophic support.
Diabetic polyneuropathy (DPN) is the most common late complication of diabetes and shows an increasing prevalence. Like other diabetes complications, DPN has been ascribed to hyperglycemia and subsequent metabolic abnormalities, particularly oxidative stress. Although other factors contribute to its development, such as insulin and C-peptide deficiencies and consequent deprivation of neurotrophic support (1), these have received less attention. Several investigators have reported neuronal apoptosis in relatively acute (2- to 3-month) streptozotocin-induced diabetes (STZ-D) in rats, sometimes exceeding 30% of dorsal root ganglion (DRG) cells as a contributing factor to DPN (2eC4), while others have reported no significant neuronal loss in DRGs even after 12 months of STZ-D (5,6). The validity of major programmed cell death can be disputed, since the magnitude of reported apoptotic neuronal death does not correlate with a corresponding loss of axonal extensions in the peripheral nerve of the STZ-D rat. In contrast to the STZ-D rat, the spontaneously type 1 diabetic BB/Wor rat develops progressive sensory nerve fiber loss (7), as in humans, which therefore could reflect loss of parent DRG cells.
Apoptotic phenomena occur in pancreatic -cells in type 1 diabetes (8), in diabetic retinopathy (9), and in hippocampal neurons in primary diabetic encephalopathy (10,11). Programmed cell death is believed to result from hyperglycemia-induced mitochondrial dysfunction (4,12) and depletion of antiapoptotic trophic factors like IGFs, insulin, and C-peptide and downregulation of their receptors (13,14). Apoptogenic stresses are mediated via increased expression of proapoptotic proteins such as Bax, oxidative DNA damage, activation of death receptors, and various upstream caspases inducing activation of caspase-3 as well as noncaspase-dependent apoptotic stressors (11). Proapoptotic activities are counteracted by stress-induced intrinsic antiapoptotic proteins, such as mitochondrial Bcl-xl, the DNA repair enzyme poly (ADP-ribose) polymerase (PARP) (5), and heat shock proteins (HSPs) (15,16).
Here, we examined subacute (4-month) spontaneously diabetic BB/Wor rats, a close model of human type 1 diabetes (17), with respect to sensory nerve functions, DRG and sural nerve morphometry and morphology, expression of active caspase-3, pro- and antiapoptotic proteins, HSPs, and trophic factors and their receptors in DRGs. Particular attention was paid to nociceptive DRG ganglion cells and their content of substance P (SP) and calcitonin geneeCrelated peptide (CGRP), since these appear to be particularly vulnerable under diabetic conditions (18,19).
RESEARCH DESIGN AND METHODS
Pre-diabetic BB/Wor rats and age-matched nondiabetes-prone BB rats were obtained from Biomedical Research Models, Worcester, Massachusetts. All rats had free access to water and rat diet. Body weight, urine volume, and glucosuria (Keto-Diastix; Bayer, Elkhart, IN) were monitored daily to ascertain onset of diabetes and for titration of daily insulin doses. After onset of diabetes, diabetic rats received titrated doses (0.5eC3.0 units/day) of protamine zinc insulin (Novo Nordisk, Princeton, NJ) to maintain blood glucose levels at 25 mmol/l glucose and to prevent ketoacidosis. Blood glucose levels were measured biweekly. Animals were cared for in accordance with guidelines of the Animal Investigation Committee, Wayne State University, and those of the National Institutes of Health (publ. no. 85-23, 1995).
Measurement of plasma insulin level.
Insulin levels were measured in plasma obtained at the time the rats were killed, 20 h after the last insulin injection, using an enzyme-linked immunosorbent assay kit (Linco Research, St. Charles, MO).
Electrophysiological recordings.
Rats were anesthetized with intraperitoneal pentobarbital sodium (50 mg/kg body wt). Rectal temperature was maintained at 36eC37°C by a heating pad and monitored by a rectal probe. Sensory nerve conduction velocity was recorded in the left hind limb as previously described (18).
Thermal plantar test.
Latencies of hind-paw withdrawal to thermal stimulation (42°C; 152 mW/cm2) was measured monthly (UGO Biological Research, Comerio, Italy) (18). The time from heat source activation to the animal’s self-withdrawal in seconds was measured six times in alternating hind paws. The mean of these measurements was calculated and used as the measure of the withdrawal latency.
Tissue collection.
Four months after onset of diabetes, animals were anesthetized. Three control and BB/Wor rats were perfused with 500 ml of 4% paraformaldehyde fixative. The left L5 DRGs were postfixed in situ for 10 min with 1% cacodylate-buffered (pH 7.4) 2.5% glutaraldehyde then excised and immersed in the same fixative for 2 h at 4°C. They were postfixed in 1% cacodylate-buffered (pH 7.4) osmium tetroxide, dehydrated, and embedded in Epon for morphologic and morphometric analyses. DRGs of the right side were fixed by immersion in 4% paraformaldehyde in 0.1 mol/l phosphate buffer (pH 7.4) overnight at 4°C, rinsed in PBS, dehydrated, immersed in xylene, and embedded in paraffin.
The right sural nerve was dissected from five diabetic and five control rats and fixed in 1% cacodylate-buffered (pH 7.4) 2.5% glutaraldehyde, dehydrated, and embedded in Epon for morphometric analysis as previously described (20,21). DRGs and sciatic nerves were collected from five diabetic and five control rats for protein extraction. Tissues were snap frozen in liquid nitrogen and kept in eC80°C until use.
Morphometry.
Morphometric analyses of DRG neurons were performed on toluidine-blueeCstained step sections (0.5 e蘭 thick; 40 e蘭 apart) using a video image analysis system (Image-Pro Plus 3.0; Media Cybernetics, Silver Spring, MD). L5 ganglia were sectioned, 35eC39 sections per ganglia. Neurons with distinguishable nuclei were counted and their area measured. Diameters of neuronal nuclei were measured and averaged. Number of neurons per DRG was calculated as the sum of numbers of neurons per section x 40/mean nuclear diameter.
Semithin (0.5-e蘭) cross-sections of Epon-embedded sural nerves were stained with toluidine-blue for light microscopic morphometric analysis using a computerized image analysis system (Image-1; Universal Imaging, West Chester, PA). This system is programmed to assess the total complement of sural nerve myelinated fibers and provides the following parameters: fasicular area (e蘭2), number of fibers (#), fiber density (#/mm2), mean fiber area (e蘭2), mean axonal area (e蘭2), mean myelin area (e蘭2), axon-to-myelin ratio, index of circularity, and fiber occupancy rate (in percent) as previously described (20,21).
For morphometric analyses of unmyelinated fibers, five animals per group were examined. Ultrathin cross-sections of sural nerves were obtained with the aid of an LKB ultramicrotome (Marviac Limited, Halifax, Canada) and stained with uranyl acetate and lead citrate. They were examined in a Zeiss EM 900 transmission electron microscope (Carl Zeiss, Oberkochen, Germany). Systematically selected frames representing 40eC50% of the sural nerve cross-sectional area were obtained. Photographs were enlarged 10,000 times, scanned, and downloaded to the computerized image analysis system. The following morphometric parameters of unmyelinated fibers were obtained: unmyelinated fiber number, fiber density (#/mm2), mean fiber size (e蘭2), axon numbers per Schwann cell unit and the frequencies of collagen pockets, denervated Schwann cell profiles, type 2 axon-Schwann cell relationship, and regenerating C-fibers (19,21).
Morphological changes.
Semithin (0.5-e蘭) toluidine-blueeCstained sections of DRGs were examined for margination of nuclear chromatin, apoptotic bodies, other degenerative changes, and nodules of Nageotte as an indication of neuronal loss.
Immunohistochemistry for SP, CGRP, HSP27, HSP70, and active caspase-3 in lumbar DRG.
Six-micrometer-thick sections were immunostained using an avidin biotin complex kit (Vector Laboratories, Burlingame, CA). Antibodies used are listed in Table 1. Quantifications of positive neurons were performed using the same image analysis system as above. Images of three serially sectioned DRG (60 e蘭 apart) were captured and assessed using a binary scale. In each ganglion, 200eC300 ganglion cells with visible nuclei were captured. The number and areas of SP- and CGRP-positive neurons were determined in each section. SP- and CGRP-positive neurons per ganglion were calculated as neurons per ganglion times positive neurons per section divided by total neurons per section. The frequencies of HSP27- and HSP70-positive neurons and caspase-3eCpositive neurons and neuronal nuclei were calculated from the same serial sections.
Measurement of SP and CGRP contents of DRGs.
Enzyme immunometric assays were used to assess SP (Cayman Chemical, Ann Arbor, MI) and CGRP (SPIbio, Massy Cedex, France) in DRGs. Results were expressed as picograms per ganglion (18).
Western blotting.
DRGs were lysed in detergent lysis buffer (50 mmol/l Tris-HCl, pH 7.4, 150 mmol/l NaC1, 1 mmol/l EDTA, 1% Triton X-100, 1 mmol/l phenylmethylsulfonyl fluoride, 1 e蘥/ml leupeptin, and 1 e蘥/ml aprotinin). The lysates were centrifuged at 14,000 rpm for 20 min at 4°C, and protein concentrations were measured using bicinchoninic acid protein assay reagent (Pierce, Rockford, IL) with BSA as standard. Ten to 40 e蘥 was separated by 7.5eC15% SDS-PAGE and transferred to polyvinylidine fluoride membranes (Bio-Rad, Hercules, CA). Membranes were blocked with Tween-20-TriseCbuffered saline (10-mmol/l Tris-HCl, pH 7.5, 100 mmol/l NaC1, and 0.1% Tween 20) containing 5% nonfat dry milk (Bio-Rad) before incubation with primary antibodies (Table 1). Antigen detection was performed using chemiluminescence (Amersham Pharmacia Biotech, Piscataway, NJ) with horseradish peroxidaseeCconjugated secondary antibodies. Membranes were exposed to Biomax film (Kodak, Rochester, NY). Images were scanned and densities determined by the Bio-Rad Fluoro-S multiimager (Bio-Rad). Expression of proteins was corrected for by actin density, and expression in control animals was arbitrarily set to 1.0.
Nerve growth factor in sciatic nerve.
Nerve growth factor (NGF) content was determined with a quantitative two-site enzyme immunoassay (Emax Immunoassay System; Promega, Madison, WI). Assays were performed according to manufacturers’ protocol. Preweighed (50 mg) sciatic nerve segments were homogenized in lysis buffer (137 mmol/l NaC1, 20 mmol/l Tris HCl, pH 8.0, 1% NP40, 10% glycerol, 1 mmol/l phenylmethylsulfonyl fluoride, 10 e蘥/ml aprotinin, 1 e蘥/ml leupeptin, and 0.5 mmol/l sodium vanadate) and centrifuged (14,000 rpm for 20 min). The supernatants were loaded on MaxiSorp 96-well plates (Nalge Nunc International, Rochester, NY), and measurements were performed as previously described (18,19). Results were expressed as picograms per milligram protein.
Statistical analysis.
All values are expressed as means ± SD. Significance of differences was analyzed by ANOVA. Group differences were assessed by Scheffes test. Significance was defined as a P value <0.05. All analyses were performed by personnel unaware of the animal identities.
RESULTS
Clinical findings.
At 4 months, BB/Wor rats showed severe hyperglycemia (control rats [n = 8]: 5.0 ± 0.4 mmol/l, diabetic rats [n = 8]: 24.4 ± 1.9 mmol/l; P < 0.001) and significant reductions in body weight (control rats [n = 8]: 458.1 ± 16.2 g, diabetic rats [n = 8]: 373.8 ± 16.4 g; P < 0.001). Serum insulin level was decreased in diabetic rats (control rats [n = 8]: 2.39 ± 0.28 ng/ml, diabetic rats [n = 8]: 0.67 ± 0.26 ng/ml; P < 0.001).
Functional data.
Sensory nerve conduction velocity decreased progressively from 91% (control rats [n = 8]: 39.8 ± 0.9 m/s, diabetic rats [n = 8]: 36.2 ± 1.2 m/s; P < 0.001) of normal at 2 months to 87% at 4 months (control rats [n = 8]: 40.9 ± 1.5 m/s, diabetic rats [n = 8]: 35.6 ± 1.6 m/s; P < 0.001). Hyperalgesia, reflecting unmyelinated fiber function, was measured as latencies of hind-paw withdrawal to thermal stimuli. Diabetic rats showed progressive and significant decreases in the latencies to thermal stimuli that reached 11.9 ± 2.7 s (n = 8) at 4 months compared with 22.7 ± 2.2 s in control rats (n = 8; P < 0.001).
Morphometry of DRG.
Neuronal size distribution of DRGs showed a shift toward smaller sizes in diabetic rats (Fig. 1A), consistent with a reduced mean neuronal size in diabetic rats (P < 0.05; Table 2). Although there was 14.7% fewer DRG neurons in diabetic rats, this did not reach statistical significance (Table 2). SP- and CGRP-positive neurons showed small shifts toward smaller sizes (Figs. 1B and C); however, the number of SP and CGRP neurons were not significantly altered (Table 2) in diabetic rats. Mean SP neuronal size was not significantly changed, whereas mean CGRP neuronal size was decreased by 15.6% (P < 0.05) in diabetic rats (Table 2).
Morphological changes.
Approximately 1,000 DRG neurons per ganglion were examined. The vast majority of ganglion cells showed normal morphology (Fig. 2A). No chromatin margination or apoptotic bodies were observed in diabetic or control rats. Instead, subplasmolemmal small vacuoles were observed in 10.5 ± 2.1 and 10.9 ± 1.7% of neurons (Fig. 2B) in control and diabetic rats, respectively. These coalesced to form larger cytoplasmic vacuoles in 0.31 ± 0.13% of neurons in diabetic rats (Fig. 2C) compared with 0.03 ± 0.06% (P < 0.05) in control rats. Nodules of Nageotte (Fig. 2D) were equally common in control and diabetic rats (0.17 ± 0.06 vs. 0.19 ± 0.13%). The structural abnormalities are consistent with hydropic degeneration.
Sural nerve morphometry.
The results of myelinated and unmyelinated fiber morphometry of sural nerves are given in Table 3. Myelinated fibers in diabetic rats showed a 15.6% fiber loss (P < 0.01) and decreased mean axonal (P < 0.05) and fiber (P < 0.005) size, whereas mean myelinated area was unchanged. The axonal atrophy was reflected by decreased index of circularity (P < 0.05).
Unmyelinated fiber morphometry of diabetic rats revealed a 28.5% fiber loss (P < 0.001), decreased fiber density (P < 0.05), axonal atrophy (P < 0.05), and decreased number of axons per Schwann cell unit (P < 0.05). These changes were accompanied by increased frequencies of denervated Schwann cell profiles (P < 0.005), collagen pockets (P < 0.05), type 2 Schwann cell/axon relationships (P < 0.001), and regenerating fibers (P < 0.001) in diabetic rats.
Expression of active caspase-3 and caspase 3eCpositive neurons.
Western blots for active caspase-3 showed increased (P < 0.05) levels in diabetic DRGs (Fig. 3A). Immunohistochemically, the frequencies of caspase-3eCpositive DRG neurons showing cytoplasmic staining (Fig. 4A) and nuclear staining (Fig. 4B) were increased (P < 0.001 and P < 0.05, respectively) in diabetic rats.
Expression of pro- and antiapoptotic elements.
Proapoptotic Bax was increased 1.9-fold (P < 0.005) in diabetic rats (Fig. 3B). This was associated with a 1.6-fold (P < 0.05) increase in antiapoptotic Bcl-xl in diabetic ganglion cells (Fig. 3C). The expression of the low-affinity NGF receptor (NGF p75R), Fas, PARP, cleaved PARP, or caspase-12, reflective of endoplasmic reticulum dysfunction, were not altered (Figs. 3DeCG).
Neurotrophic factors.
The NGF content in the sciatic nerve was significantly decreased (P < 0.05) in diabetic rats (Fig. 5H). The high-affinity NGF receptor (TrkA) expression in diabetic DRGs was significantly decreased (P < 0.05; Fig. 5E), whereas the IGF-1 receptor and insulin receptor expressions in DRGs were not altered (Figs. 5F and G).
SP and CGRP contents in DRGs.
SP and CGRP contents in DRGs were not significantly changed in diabetic rats (Table 2).
HSPs in DRGs.
HSP27 protein expression was significantly (P < 0.05) increased in diabetic DRGs (Fig. 5A). This change correlated with an increased frequency (P < 0.05) of immunohistochemically positive neurons (Fig. 6A). HSP27-positive staining was observed in the cytoplasm of neurons and axons of all sizes. The expression of inducible HSP70 was not altered in diabetic DRGs (Fig. 5B). Immunostaining of HSP70 in DRGs showed localization mainly to Schwann cells, satellite cells, and small neurons (Fig. 6B). Although the percentage of HSP70-positive neurons was low, it was significantly increased (P < 0.05) in diabetic DRGs. Constitutively expressed HSC70 or HSP40 were not altered (Figs. 5C and D).
DISCUSSION
Human DPN shows progressive loss of myelinated and unmyelinated sensory fibers in peripheral nerves, accompanied by degeneration and loss of parent DRG neurons, and is classified as a peripheral axonopathy of dying-back type (1,7,22eC24). Hyperglycemia with consequent perturbations of the polyol pathway, nerve hypoxemia, and oxidative stress have been suggested as the main culprits (1). In type 1 DPN, impaired insulin and C-peptide signaling plays contributing roles, causing perturbed gene regulation of neurotrophic factors and their receptors and aberrations of the expression and posttranslational modifications of neuroskeletal and adhesive proteins (1,18,19,24,25). It has been claimed that apoptosis of sensory ganglion cells is a prominent component in the pathogenesis of DPN in STZ-D rats (2eC4,12).
Using the spontaneously type 1 diabetic BB/Wor rat we demonstrate here 1) increased stainability and upregulation of active caspase-3 and proapoptotic Bax in diabetic DRGs, 2) that these apoptotic stresses are accompanied by the induction of antiapoptotic Bcl-xl and survival promoting HSP27 and HSP70, 3) that NGF content in sciatic nerve and high-affinity NGF receptor TrkA expression in diabetic DRGs are decreased, 4) that morphometric analyses of sural nerve reveal myelinated and unmyelinated fiber loss and axonal atrophy in diabetic rats, and 5) that morphometric and morphologic examinations of DRGs show no neuronal loss or qualitative changes indicative of apoptosis. Instead, DRG neurons show progressive hydropic damage similar to that described in human DPN (22).
Caspase-3 is the common final executing caspase and is activated by several upstream apoptotic pathways. Here, we demonstrate increased expression of active caspase-3 as well as increased stainability in neuronal cytoplasm and nuclei in diabetic rats. These findings are in agreement with previous data (2,5,6) and were accompanied by increased expression of Bax, indicative of mitochondrial dysfunction. On the other hand, these apoptotic stressors were associated with upregulation of antiapoptotic Bcl-xl and HSP27 as well as an increased number of neurons staining positive for HSP70.
HSPs are proteins induced by thermal stimuli and act as chaperones to support folding of proteins. HSP27 and HSP70 have been widely investigated, and their protective effect against neuronal death has been repeatedly reported (15,16,26eC30). HSP27 is constitutively expressed and can be induced not only by thermal stimuli but also by trophic factor withdrawal or by apoptotic stress (26). HSP27 interacts with cytochrome c, blocking its interaction with Apaf-1, procaspase 9, and downstream activation of caspase-3 (26), and it inhibits Fas-induced caspase-independent apoptosis by interacting with Daxx (30). However, Fas was not increased in DRGs in this study, suggesting that caspase-independent mechanisms are not involved. Inducible HSP70 is a survival element (28,29) induced by several stresses (31). In this study, we found localization of HSP70 in Schwann cells and small neurons. Although its expression in DRGs was not changed, immunohistochemistry revealed increased HSP70 positivity in small DRG neurons. HSP70 may therefore be increased in the more susceptible small nociceptive neurons under diabetic conditions. In keeping with the present findings, Guzhova et al. (32) reported that HSP70 may be exported from glia cells and that extracellular HSP70 has protective effects on neurons.
Taken together with the morphometric and morphologic data, which failed to demonstrate significant neuronal loss and qualitative changes suggestive of ongoing apoptosis, it appears that apoptotic stresses are induced in DRGs but that the final execution of DRG neuronal apoptosis is prevented by the induction of counterregulatory elements such as Bcl-xl and HSPs. This is supported by the findings that the common death substrate protein PARP and its cleavage product, indicative of DNA damage, were unaltered in diabetic DRGs.
These findings differ from those previously reported in the hippocampus of the same rat model (11), in which activation of caspase-3 and increased expression of Bax were not counteracted by induction of Bcl-xl. Futhermore, activation of FAS and the death-receptor NGF-p75R as well as increased expression of cleaved PARP reported in hippocampus could not be demonstrated in DRGs. The apoptotic phenomena seen in hippocampus were associated with significant neuronal loss (11), which therefore suggest that diabetes induces a more severe apoptotic stress in central neuronal populations than in peripheral DRG neurons.
Instead, DRG neurons showed atrophy and vacuolar cytoplasmic degeneration, characteristic of hydropic degeneration. The frequency of nodules of Nageotte, signifying ganglion cell loss, was not increased in diabetic rats. The mechanisms underlying these changes are not known. However, it is likely that impaired synthesis of neuroskeletal proteins previously demonstrated in this model (33) may account for not only axonal atrophy but also for the atrophy of neuronal perikaria as demonstrated here (24). The vacuolar changes are likely to reflect impaired neuronal energy metabolism and perturbed sodium handling possibly via impaired Na+/K+-ATPase activity and/or decreased DRG blood flow (34) and may therefore be analogous to nodal axonal swellings seen in peripheral nerve fibers in DPN (35). These constructs are consistent with impaired insulin/C-peptide action affecting Na+/K+-ATPase activity and resulting in downregulation of NGF and suppression of its high-affinity TrkA receptor as demonstrated here (18,19,36eC38).
Abnormalities in insulin and NGF signaling but not in that of IGF-1 may account for the greater susceptibility of C-fibers and their parent NGF-dependent neurons in DRGs. This is supported by the findings in the isohyperglycemic and hyperinsulinemic type 2 counterpart to this type 1 diabetes model, in which perturbations of NGF and its high-affinity receptor are milder and associated with less severe C-fiber pathology (19).
Increased hyperalgesia, as reported here and previously (18,19,21), has been associated with increased spontaneous firing of nociceptive neurons (39) due to upregulation of 3 and tetrodotoxin-resistant voltage-gated Na+ channels in small nociceptive and A DRG neurons (40,41). This will inevitably result in increased Na+ influx as demonstrated by increased Na+ current densities and a shift in steady-state inactivation in diabetic dorsal root ganglia (41). Coupled with perturbed Na+/K+-ATPase activity, these changes may not only account for the hyperexcitability of nociceptive DRGs but also for the observed progressive hydropic degenerative changes. Thermal hypoalgesia is observed in humans and STZ-D rats with DPN. In STZ-D rats, this is proceeded by a short period of hyperalgesia (42eC45) analogous to a transient period of hyperalgesia in BB/Wor rats (18,19). The transient hyperalgesia most likely corresponds to anatomically intact hyperexitable C-fibers, whereas the ensuing hypoalgesia probably reflects the progressive denervation by C-fibers due to the dying-back process.
In summary, in the present study we could not confirm previous reports on high apoptotic activity and cell death in DRGs. Instead, the present data are consistent with those reported by Cheng and Zochodne (5), who failed to show loss of DRG neurons in 12-month STZ-D rats. The data are also in keeping with those reported by Burnand et al. (46), who failed to demonstrate the induction of apoptosis-related genes in acutely (8 weeks) STZ-D rats. The axonal loss and DRG neuronal atrophy reported here are consistent with an axonal dying-back phenomenon due to impaired cytoskeletal protein synthesis (6,7,33) secondary to insulin/C-peptide deficiencies and deprived neurotrophic support.
ACKNOWLEDGMENTS
This study was supported by research grants from the Thomas Foundation, which is also providing support to the postdoctoral fellowship of H.K., and the Juvenile Diabetes Research Foundation.
REFERENCES
Sima AAF: New insights into the metabolic and molecular basis for diabetic neuropathy. Cell Mol Life Sci60 :2445 eC2464,2003
Schmeichel AM, Schmetzer JD, Low PA: Oxidative injury and apoptosis of dorsal root ganglion neurons in chronic experimental diabetic neuropathy. Diabetes52 :165 eC171,2003
Russel JW, Sullivan KA, Windebank AJ, Herrmann DN, Feldman EL: Neurons undergo apoptosis in animal and cell culture models of diabetes. Neurobiol Dis6 :347 eC363,1999
Scrinivasan S, Stevens M, Wiley JW: Diabetic peripheral neuropathy: evidence for apoptosis and associated mitochondrial dysfunction. Diabetes49 :1932 eC1938,2000
Cheng C, Zochodne DW: Sensory neurons with activated caspase-3 survive long-term experimental diabetes. Diabetes52 :2363 eC2371,2003
Zochodne DW, Verge VM, Cheng C, Sun H, Johnston J: Does diabetes target ganglion neurones Progressive sensory neurone involvement in long-term experimental diabetes. Brain124 :2319 eC2334,2001
S ima AA, Bouchier M, Christensen H: Axonal atrophy in sensory nerves of the diabetic BB-Wistar rat: a possible early correlate of human diabetic neuropathy. Ann Neurol13 :264 eC272,1983
Thompson CB: Apoptosis in the pathogenesis and treatment of disease. Science267 :1456 eC1462,1995
Barber AJ, Lieth E, Khin SA, Antonetti DA, Buchanan AG, Gardner TW: Neural apoptosis in the retina during experimental and human diabetes: early onset and effect of insulin. J Clin Invest102 :783 eC791,1998
Li ZG, Zhang W, Grunberger G, Sima AA: Hippocampal neuronal apoptosis in type 1 diabetes. Brain Res946 :221 eC231,2002
Sima AAF, Li Z-G: The effect of C-peptide on cognitive dysfunction and hippocampal apoptosis in type 1 diabetes. Diabetes54 :1497 eC1505,2005
Vincent AM, Brownlee M, Russel JW: Oxidative stress and programmed cell death in diabetic neuropathy. Ann N Y Acad Sci959 :368 eC383,2002
Russel JW, Feldman EL: Insulin-like growth factor-1 prevents apoptosis in sympathetic neurons exposed to high glucose. Horm Metab Res31 :90 eC96,1999
Li Z-G, Zhang W, Sima AAF: C-peptide enhances insulin-mediated cell growth and protection against high glucose induced apoptosis in SH-SY5Y cells. Diabetes Metab Res Rev19 :375 eC385,2003
Li C-Y, Lee J-S, Ko Y-G, Kim J-I, Seo J-S: Heat shock protein 70 inhibits apoptosis downstream of cytochrome c release and upstream of caspase-3 activation. J Biol Chem275 :25665 eC25671,2000
Lewis SE, Mannion RJ, White FA, Coggeshall RE, Beggs S, Costigan M, Martin JL, Dillmann WH, Woolf CJ: A role for HSP27 in sensory neuron survival. J Neurosci19 :8945 eC8953,1999
Sima AAF: Diabetes underlies common neurological disorders (Editorial). Ann Neurol56 :459 eC461,2004
Kamiya H, Zhang W, Sima AAF: C-peptide prevents nociceptive sensory neuropathy in type 1 diabetes. Ann Neurol56 :827 eC835,2004
Kamiya H, Murakawa Y, Zhang W, Sima AAF: Unmyelinated fiber sensory neuropathy differs in type 1 and type 2 diabetes. Diabetes Metab Res Rev21 :448 eC458,2005
Sima AAF, Zhang W, Sugimoto K, Henry D, Li Z, Wahren J, Grunberger G: C-peptide prevents and improves chronic type 1 diabetic neuropathy in the BB/Wor-rat. Diabetologia44 :889 eC897,2001
Murakawa Y, Zhang W, Pierson CR, Brismar T, Ostenson CG, Efendic S, Sima AAF: Impaired glucose tolerance and insulinopenia in the GK-rat causes peripheral neuropathy. Diabetes Metab Res Rev18 :473 eC483,2002
Greenbaum D, Richardson PC, Salmon MW, Urich, H: Pathologic aberrations on six cases of diabetic neuropathy. Brain87 :201 eC214,1964
Sima AAF, Nathaniel V, Bril V, McEwen TAJ, Greene DA: Histopathological heterogeneity of neuropathy in insulin-dependent and noninsulin-dependent diabetes, and demonstration of axo-glial dysjunction in human diabetic neuropathy. J Clin Invest81 :349 eC364,1988
Scott JN, Clark AW, Zochodne DW: Neurofilament and tubulin gene expression in progressive experimental diabetes: failure of synthesis and export by neurons. Brain122 :2109 eC2117,1999
Sima AAF, Zhang W, Li Z-G, Murakawa Y, Pierson CR: Molecular alterations underlie nodal and paranodal degeneration in type 1 diabetic neuropathy and are prevented by C-peptide. Diabetes53 :1556 eC1563,2004
Benn SC, Perrelet D, Kato AC, Scholz J, Decosterd I, Mannion RJ, Bakowska JC, Woolf CJ: Hsp27 upregulation and phosphorylation is required for injured sensory and motor neuron survival. Neuron36 :45 eC56,2002
Gurbuxani S, Schmitt E, Cande C, Parcellier A, Hammann A, Daugas E, Kouranti I, Spahr C, Pance A, Kroemer G, Garrido C: Heat shock protein 70 binding inhibits the nuclear import of apoptosis-inducing factor. Oncogene22 :6669 eC6678,2003
Beere HM, Wolf BB, Cain K, Mosser DD, Mahboubi A, Kuwana T, Tailor P, Morimoto RI, Cohen GM, Green DR: Heat shock protein 70 inhibits apoptosis by preventing recruitment of procaspase-9 to the Apaf-1 apoptosome. Nat Cell Biol2 :469 eC475,2000
Ravagnan L, Gurbuxani S, Susin SA, Maisse C, Daugas E, Zamzami N, Mak T, Jaattela M, Penninger JM, Garrido C, Kroemer G: Heat shock protein 70 antagonizes apoptosis-inducing factor. Nat Cell Biol3 :839 eC843,2001
Mehlen P, Schulzeosthoff K, Arrigo AP: Small stress proteins as novel regulators of apoptosis: heat shock protein 27 blocks Fas/APO-1 and stanrosporine-induced cell death. J Biol Chem271 :16510 eC16514,1996
Yenari MA: Heat shock proteins and neuroprotection. Adv Exp Med Biol513 :281 eC299,2002
Guzhova I, Kislyakova K, Moskaliova O, Fridlanskaya I, Tytell M, Cheetham M, Margulis B: In vitro studies show that Hsp70 can be released by glia and that exogenous Hsp70 can enhance neuronal stress tolerance. Brain Res914 :66 eC73,2001
Xu G, Pierson CR, Murakawa Y, Sima AAF: Altered tubulin and neurofilament expression and impaired axonal growth in diabetic nerve regeneration. J Neuropath Exp Neurol61 :164 eC175,2002
Zochodne DW, Ho LT, Allison JA: Dorsal root ganglia microenvironment of female BB Wistar diabetic rats with mild neuropathy. J Neurol Sci127 :36 eC42,1994
Sima AAF, Brismar T: Reversible diabetic nerve dysfunction: structural correlates to electrophysiological abnormalities. Ann Neurol18 :21 eC29,1985
Reico-Pinto E, Lang FF, Ishii DN: Insulin and insulin-like growth factor II permit nerve growth factor binding and the neurite formation response in cultured neuroblastoma cells. Proc Natl Acad Sci U S A81 :2562 eC2566,1984
Fernyhough P, Diemel LT, Brewster WJ, Tomlinson DR: Deficits in sciatic nerve neuropeptide content coincide with a reduction in target tissue nerve growth factor messenger RNA in streptozotocin-diabetic rats: effects of insulin treatment. Neurosci62 :337 eC344,1994
Zhang W, Yorek M, Pierson CR, Murakawa Y, Breidenbach A, Sima AA: Human C-peptide dose dependently prevents early neuropathy in the BB/Wor-rat. Int J Exp Diabetes Res2 :187 eC193,2001
Burchiel KJ, Russell LC, Lee RP, Sima AAF: Spontaneous activity of primary afferent neurons in diabetic BB/Wistar rats. Diabetes34 :1210 eC1213,1985
Shah BS, Gonzalez I, Bramwell S, Pinnock RD, Lee K, Dixon AK: 3, a novel auxillary subunit for the voltage gated sodium channel is upregulated in sensory neurons following streptozotocin induced diabetic neuropathy in rat. Neurosci Lett309 :1 eC4,2001
Hirade M, Yasuda H, Omatsu-Kanbe M, Kikkawa R, Kitasato H: Tetrodotoxin-resistant sodium channels of dorsal root ganglion neurons are readily activated in diabetic rats. Neurosci90 :933 eC939,1999
Li F, Obrosova IG, Abatan O, Tian D, Larkin D, Stuenkel EL, Stevens MJ: Taurine replacement attenuates hyperalgesia and abnormal calcium signaling in sensory neurons of STZ-D rats. Am J Physiol Endocrinol Metab288 :E29 eCE36,2005
Cameron NE, Tuck Z, McCabe L, Cotter MA: Effect of the hydroxyl radical scavenger, dimethylthiourea, on peripheral nerve tissue perfusion, conduction velocity and nociception in experimental diabetes. Diabetologia44 :1161 eC1169,2001
Fox A, Eastwood C, Gentry C, Manning D, Urban L: Critical evaluation of the streptozotocin model of painful diabetic neuropathy in the rat. Pain81 :307 eC316,1999
Calcutt NA, Freshwater JD, Mizisin AP: Prevention of sensory disorders in diabetic Sprague-Dawley rats by aldose reductase inhibition or treatment with ciliary neurotrophic factor. Diabetologia47 :718 eC724,2004
Burnand RC, Price SA, McElhaney M, Barker D, Tomlinson DR: Expression of axotomy-inducible and apoptosis-related genes in sensory nerves of rats with experimental diabetes. Brain Res Mol Brain Res20 :235 eC240,2004(Hideki Kamiya, Weixian Zh)