Diabetic Dorsal Root Ganglion Research Articles

Modulation of Neuronal Damage in DRG by Asprosin in a High-Glucose Environment and Its Impact on miRNA181-a Expression in Diabetic DRG.

Asprosin, a hormone secreted from adipose tissue, has been implicated in the modulation of cell viability. Current studies suggest that neurological impairments are increased in individuals with obesity-linked diabetes, likely due to the presence of excess adipose tissue, but the precise molecular mechanism behind this association remains poorly understood. In this study, our hypothesis that asprosin has the potential to mitigate neuronal damage in a high glucose (HG) environment while also regulating the expression of microRNA (miRNA)-181a, which is involved in critical biological processes such as cellular survival, apoptosis, and autophagy. To investigate this, dorsal root ganglion (DRG) neurons were exposed to asprosin in a HG (45 mmol/L) environment for 24 hours, with a focus on the role of the protein kinase A (PKA) pathway. Expression of miRNA-181a was measured by using real-time polymerase chain reaction (RT-PCR) in diabetic DRG. Our findings revealed a decline in cell viability and an upregulation of apoptosis under HG conditions. However, pretreatment with asprosin in sensory neurons effectively improved cell viability and reduced apoptosis by activating the PKA pathway. Furthermore, we observed that asprosin modulated the expression of miRNA-181a in diabetic DRG. Our study demonstrates that asprosin has the potential to protect DRG neurons from HG-induced damage while influencing miRNA-181a expression in diabetic DRG. These findings provide valuable insights for the development of clinical interventions targeting neurotoxicity in diabetes, with asprosin emerging as a promising therapeutic target for managing neurological complications in affected individuals.

Metformin prevents morphine-induced apoptosis in rats with diabetic neuropathy: a possible mechanism for attenuating morphine tolerance.

Morphine is a drug of choice for the treatment of severe and chronic pain, but tolerance to the antinociceptive effect limits its use. The development of tolerance to morphine has recently been associated with neuronal apoptosis. In this study, our aim was to investigate the effects of metformin on morphine-induced neuronal apoptosis and antinociceptive tolerance in diabetic rats. Three days of cumulative dosing were administered to establish morphine tolerance in rats. The antinociceptive effects of metformin (50mg/kg) and test dose of morphine (5mg/kg) were considered at 30-min intervals by thermal antinociceptive tests. To induce diabetic neuropathy, streptozotocin (STZ, 65mg/kg) was injected intraperitoneally. ELISA kits were used to measure caspase-3, bax, and bcl-2 levels from dorsal root ganglion (DRG) tissue. Semi-quantitative scoring system was used to evaluate apoptotic cells with the the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) method. The findings suggest that co-administration of metformin with morphine to diabetic rats showed a significant increase in antinociceptive effect compared to morphine alone. The antinociceptive tests indicated that metformin significantly attenuated morphine antinociceptive tolerance in diabetic rats. In addition, metformin decreased the levels of apoptotic proteins caspase 3 and Bax in DRG neurons, while significantly increased the levels of antiapoptotic Bcl-2. Semi-quantitative scoring showed that metformin provided a significant reduction in apoptotic cell counts in diabetic rats. These data revealed that metformin demonstrated antiapoptotic activity in diabetic rat DRG neurons and attenuated morphine tolerance. The antiapoptotic activity of metformin probably plays a significant role in reducing morphine tolerance.

The Role of Long Noncoding RNA MALAT1 in Diabetic Polyneuropathy and the Impact of Its Silencing in the Dorsal Root Ganglion by a DNA/RNA Heteroduplex Oligonucleotide.

Diabetic polyneuropathy (DPN) is the most common complication of diabetes, yet its pathophysiology has not been established. Accumulating evidence suggests that long noncoding RNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) plays pivotal roles in the regulation of cell growth and survival during diabetic complications. This study aimed to investigate the impact of MALAT1 silencing in dorsal root ganglion (DRG) sensory neurons, using an α-tocopherol-conjugated DNA/RNA heteroduplex oligonucleotide (Toc-HDO), on the peripheral nervous system of diabetic mice. We identified MALAT1 upregulation in the DRG of chronic diabetic mice that suggested either a pathological change or one that might be protective, and systemic intravenous injection of Toc-HDO effectively inhibited its gene expression. However, we unexpectedly noted that this intervention paradoxically exacerbated disease with increased thermal and mechanical nociceptive thresholds, indicating further sensory loss, greater sciatic-tibial nerve conduction slowing, and additional declines of intraepidermal nerve fiber density in the hind paw footpads. Serine/arginine-rich splicing factors, which are involved in pre-mRNA splicing by interacting with MALAT1, reside in nuclear speckles in wild-type and diabetic DRG neurons; MALAT1 silencing was associated with their disruption. The findings provide evidence for an important role that MALAT1 plays in DPN, suggesting neuroprotection and regulation of pre-mRNA splicing in nuclear speckles. This is also the first example in which a systemically delivered nucleotide therapy had a direct impact on DRG diabetic neurons and their axons.

Taurine ameliorates axonal damage in sciatic nerve of diabetic rats and high glucose exposed DRG neuron by PI3K/Akt/mTOR-dependent pathway.

Diabetic peripheral neuropathy (DPN) is a common complication of diabetes and axonopathy is its main pathological feature. Previous studies suggested an advantage of taurine against diabetes. However, there are few reports which study the effect of taurine against axonopathy. In this study, we confirmed that taurine significantly decreased blood glucose level, mitigated insulin resistance and improved dysfunctional nerve conduction in diabetic rats. Taurine corrected damaged axonal morphology of sciatic nerve in diabetic rats and induced axon outgrowth of Dorsal root ganglion (DRG) neurons exposed to high glucose. Taurine up-regulated phosphorylation levels of PI3K, Akt, and mTOR in sciatic nerve of diabetic rats and DRG neurons exposed to high glucose. However, Akt and mTOR inhibitors (MK-2206 and Rapamycin) blocked the effect of taurine on improving axonal damage. These results indicate that taurine ameliorates axonal damage in sciatic nerve of diabetic rats by activating PI3K/Akt/mTOR signal pathway. Our findings provide taurine as a potential candidate for axonopathy and a new evidence for elucidating protective mechanism of taurine on DPN.

Satellite glia as a critical component of diabetic neuropathy: Role of lipocalin-2 and pyruvate dehydrogenase kinase-2 axis in the dorsal root ganglion.

Diabetic peripheral neuropathy (DPN) is a common complication of uncontrolled diabetes. The pathogenesis of DPN is associated with chronic inflammation in dorsal root ganglion (DRG), eventually causing structural and functional changes. Studies on DPN have primarily focused on neuronal component, and there is limited knowledge about the role of satellite glial cells (SGCs), although they completely enclose neuronal soma in DRG. Lipocalin-2 (LCN2) is a pro-inflammatory acute-phase protein found in high levels in diverse neuroinflammatory and metabolic disorders. In diabetic DRG, the expression of LCN2 was increased exclusively in the SGCs. This upregulation of LCN2 in SGCs correlated with increased inflammatory responses in DRG and sciatic nerve. Furthermore, diabetes-induced inflammation and morphological changes in DRG, as well as sciatic nerve, were attenuated in Lcn2 knockout (KO) mice. Lcn2 gene ablation also ameliorated neuropathy phenotype as determined by nerve conduction velocity and intraepidermal nerve fiber density. Mechanistically, studies using specific gene KO mice, adenovirus-mediated gene overexpression strategy, and primary cultures of DRG SGCs and neurons have demonstrated that LCN2 enhances the expression of mitochondrial gate-keeping regulator pyruvate dehydrogenase kinase-2 (PDK2) through PPARβ/δ, thereby inhibiting pyruvate dehydrogenase activity and increasing production of glycolytic end product lactic acid in DRG SGCs and neurons of diabetic mice. Collectively, our findings reveal a crucial role of glial LCN2-PPARβ/δ-PDK2-lactic acid axis in progression of DPN. Our results establish a link between pro-inflammatory LCN2 and glycolytic PDK2 in DRG SGCs and neurons and propose a novel glia-based mechanism and drug target for therapy of DPN. MAIN POINTS: Diabetes upregulates LCN2 in satellite glia, which in turn increases pyruvate dehydrogenase kinase-2 (PDK2) expression and lactic acid production in dorsal root ganglia (DRG). Glial LCN2-PDK2-lactic acid axis in DRG plays a crucial role in the pathogenesis of diabetic neuropathy.

WenTong HuoXue Cream Can Inhibit the Reduction of the Pain-Related Molecule PLC-β3 in the Dorsal Root Ganglion of a Rat Model of Diabetic Peripheral Neuropathy.

WenTong HuoXue Cream (WTHX-Cream) has been shown to effectively alleviate clinical symptoms of diabetic peripheral neuropathy (DPN). This study investigated the gene and protein expression of the pain-related molecule PLC-β3 in the dorsal root ganglion (DRG) of DPN rats. 88 specific pathogen-free male Wistar rats were randomly divided into placebo (10 rats) and DPN model (78 rats) groups, and the 78 model rats were used to establish the DPN model by intraperitoneal injection of streptozotocin and were then fed a high-fat diet for 8 weeks. These rats were randomly divided into the model group, the high-, medium-, and low-dose WTHX-Cream + metformin groups, the metformin group, the capsaicin cream group, and the capsaicin cream + metformin group. After 4 weeks of continuous drug administration, the blood glucose, body weight, behavioral indexes, and sciatic nerve conduction velocity were measured. The pathological structure of the DRG and the sciatic nerve were observed. PLC-β3 mRNA and protein levels in the DRG of rats were measured. Compared with the model group, the high-dose WTHX-Cream group showed increased sciatic nerve conduction velocity, improved sciatic nerve morphological changes, and increased expression of PLC-β3 mRNA and protein in the DRG. This study showed that WTHX-Cream improves hyperalgesia symptoms of DPN by inhibiting the reduction of PLC-β3 mRNA and protein expression in the diabetic DRG of DPN rats.

Pyruvate Dehydrogenase Kinase-mediated Glycolytic Metabolic Shift in the Dorsal Root Ganglion Drives Painful Diabetic Neuropathy

The dorsal root ganglion (DRG) is a highly vulnerable site in diabetic neuropathy. Under diabetic conditions, the DRG is subjected to tissue ischemia or lower ambient oxygen tension that leads to aberrant metabolic functions. Metabolic dysfunctions have been documented to play a crucial role in the pathogenesis of diverse pain hypersensitivities. However, the contribution of diabetes-induced metabolic dysfunctions in the DRG to the pathogenesis of painful diabetic neuropathy remains ill-explored. In this study, we report that pyruvate dehydrogenase kinases (PDK2 and PDK4), key regulatory enzymes in glucose metabolism, mediate glycolytic metabolic shift in the DRG leading to painful diabetic neuropathy. Streptozotocin-induced diabetes substantially enhanced the expression and activity of the PDKs in the DRG, and the genetic ablation of Pdk2 and Pdk4 attenuated the hyperglycemia-induced pain hypersensitivity. Mechanistically, Pdk2/4 deficiency inhibited the diabetes-induced lactate surge, expression of pain-related ion channels, activation of satellite glial cells, and infiltration of macrophages in the DRG, in addition to reducing central sensitization and neuroinflammation hallmarks in the spinal cord, which probably accounts for the attenuated pain hypersensitivity. Pdk2/4-deficient mice were partly resistant to the diabetes-induced loss of peripheral nerve structure and function. Furthermore, in the experiments using DRG neuron cultures, lactic acid treatment enhanced the expression of the ion channels and compromised cell viability. Finally, the pharmacological inhibition of DRG PDKs or lactic acid production substantially attenuated diabetes-induced pain hypersensitivity. Taken together, PDK2/4 induction and the subsequent lactate surge induce the metabolic shift in the diabetic DRG, thereby contributing to the pathogenesis of painful diabetic neuropathy.

Diabetes enhances oxidative stress-induced TRPM2 channel activity and its control by N-acetylcysteine in rat dorsal root ganglion and brain.

N-acetylcysteine (NAC) is a sulfhydryl donor antioxidant that contributes to the regeneration of glutathione (GSH) and also scavengers via a direct reaction with free oxygen radicals. Recently, we observed a modulatory role of NAC on GSH-depleted dorsal root ganglion (DRG) cells in rats. NAC may have a protective role on oxidative stress and calcium influx through regulation of the TRPM2 channel in diabetic neurons. Therefore, we investigated the effects of NAC on DRG TRPM2 channel currents and brain oxidative stress in streptozotocin (STZ)-induced diabetic rats. Thirty-six rats divided into four groups: control, STZ, NAC and STZ + NAC. Diabetes was induced in the STZ and STZ + NAC groups by intraperitoneal STZ (65 mg/kg) administration. After the induction of diabetes, rats in the NAC and STZ + NAC groups received NAC (150 mg/kg) via gastric gavage. After 2 weeks, DRG neurons and the brain cortex were freshly isolated from rats. In whole-cell patch clamp experiments, TRPM2 currents in the DRG following diabetes induction with STZ were gated by H2O2. TRPM2 channel current densities in the DRG and lipid peroxidation levels in the DRG and brain were higher in the STZ groups than in controls; however, brain GSH, GSH peroxidase (GSH-Px), vitamin C and vitamin E concentrations and DRG GSH-Px activity were decreased by diabetes. STZ + H2O2-induced TRPM2 gating was totally inhibited by NAC and partially inhibited by N-(p-amylcinnamoyl) anthranilic acid (ACA) and 2-aminoethyl diphenylborinate (2-APB). GSH-Px activity and lipid peroxidation levels were also attenuated by NAC treatment. In conclusion, we observed a modulatory role of NAC on oxidative stress and Ca(2+) entry through the TRPM2 channel in the diabetic DRG and brain. Since excessive oxidative stress and overload Ca(2+) entry are common features of neuropathic pain, our findings are relevant to the etiology and treatment of pain neuropathology in DRG neurons.

Comparative evaluation of in vitro and in vivo high glucose-induced alterations in voltage-gated tetrodotoxin-resistant sodium channel: Effects attenuated by sodium channel blockers

Glucose uptake in neurons depends on their cellular/physiological activity and the extracellular concentration of glucose around the cell. High concentration of extra-cellular glucose, as under hyperglycemic conditions or pathological condition in diabetes, may persist for extended periods of time in neurons and trigger cellular damage by altering voltage-gated sodium channels (VGSCs), the exact mechanism of which remains unclear. Therefore, we hypothesized that high glucose may directly affect kinetics of the VGSCs in the dorsal root ganglion (DRG) neurons. DRG neurons were exposed to normal glucose (NG: 5.5mM) and high glucose (HG: 30mM) for 24h. In another set of experiments, diabetic DRG neurons were also isolated from streptozotocin-induced diabetic rats. Effects of sodium channel blockers on nociceptive parameters and tetrodotoxin-resistant (TTX-R) Na+ channel kinetics were elucidated by whole-cell patch-clamp in HG exposure and diabetes-induced rat DRG neurons. HG exposure and diabetes-induced DRG neurons demonstrated significant increase in TTX-R Na+ current (INa) densities in comparison to the control. Both HG-exposed and diabetic DRG neurons demonstrated similar biophysical characteristics of INa. Lidocaine and tetracaine significantly decreased TTX-R INa density in a concentration- and voltage-dependent manner. Steady-state fast inactivation of INa was shifted in the hyperpolarizing direction whereas voltage-dependent activation was shifted in the rightward direction. Diabetic rats treated with lidocaine and tetracaine (3mg/kg, i.p.) significantly improved thermal hyperalgesia, mechanical allodynia and motor nerve conduction velocity with a significant inhibition of TTX-R INa density as compared to the diabetic control. These results suggest that HG exposure increases the TTX-R Na+ channel activity sensitive to Na+ channel blockers, lidocaine and tetracaine.

Rufinamide Improves Functional and Behavioral Deficits <i>via</i> Blockade of Tetrodotoxin-Resistant Sodium Channels in Diabetic Neuropathy

Rufinamide is a structurally novel, antiepileptic drug approved for the treatment of Lennox-Gastaut syndrome. Its mechanism of action involves inhibition of voltage-gated Na+ channels (VGSCs) with possible membrane-stabilizing effects. VGSCs play a significant role in the pathogenesis of neuropathic pain. Therefore, we investigated the effects of rufinamide on tetrodotoxin-resistant sodium current (TTX-R I(Na)) in acutely dissociated rat dorsal root ganglion (DRG) neurons isolated from streptozotocin-induced diabetic rats by using whole-cell voltage-clamp configuration. In addition, the functional and behavioural nociceptive parameters were evaluated to assess its potential in diabetic neuropathy. Diabetic rats demonstrated the mechanical allodynia and thermal hyperalgesia with reduced nerve perfusion and conduction velocity as compared to control. Rufinamide treatments (3 and 10 mg/kg) significantly improved these functional and nociceptive deficits. Diabetic rat DRG neurons exhibited increased TTX-R I(Na) density as compared to control. The voltage-dependent activation and steady-state inactivation curves for TTX-R I(Na) in DRG neurons from diabetic rats were shifted negatively as compared to control. Rufinamide treatments significantly blocked the TTX-R Na+ channel activity as evident from significant reduction in I(Na) density and hyperpolarizing shift in activation and inactivation curves as compared to diabetic control. This suggests that rufinamide acts on TTX-R Na+ channels, reduces channel activity and attenuates nerve functional and behavioral parameters in diabetic rats. Altogether, these results indicate therapeutic potential of rufinamide in the treatment of diabetic neuropathy.

PGC-1α regulation of mitochondrial degeneration in experimental diabetic neuropathy

Mitochondrial degeneration is considered to play an important role in the development of diabetic peripheral neuropathy in humans. Mitochondrial degeneration and the corresponding protein regulation associated with the degeneration were studied in an animal model of diabetic neuropathy. PGC-1α and its-regulated transcription factors including TFAM and NRF1, which are master regulators of mitochondrial biogenesis, are significantly downregulated in streptozotocin diabetic dorsal root ganglion (DRG) neurons. Diabetic mice develop peripheral neuropathy, loss of mitochondria, decreased mitochondrial DNA content and increased protein oxidation. Importantly, this phenotype is exacerbated in PGC-1α (−/−) diabetic mice, which develop a more severe neuropathy with reduced mitochondrial DNA and a further increase in protein oxidation. PGC-1α (−/−) diabetic mice develop an increase in total cholesterol and triglycerides, and a decrease in TFAM and NRF1 protein levels. Loss of PGC-1α causes severe mitochondrial degeneration with vacuolization in DRG neurons, coupled with reduced state 3 and 4 respiration, reduced expression of oxidative stress response genes and an increase in protein oxidation. In contrast, overexpression of PGC-1α in cultured adult mouse neurons prevents oxidative stress associated with increased glucose levels. The study provides new insights into the role of PGC-1α in mitochondrial regeneration in peripheral neurons and suggests that therapeutic modulation of PGC-1α function may be an attractive approach for treatment of diabetic neuropathy.

Gastrodin inhibits allodynia and hyperalgesia in painful diabetic neuropathy rats by decreasing excitability of nociceptive primary sensory neurons.

Painful diabetic neuropathy (PDN) is a common complication of diabetes mellitus and adversely affects the patients’ quality of life. Evidence has accumulated that PDN is associated with hyperexcitability of peripheral nociceptive primary sensory neurons. However, the precise cellular mechanism underlying PDN remains elusive. This may result in the lacking of effective therapies for the treatment of PDN. The phenolic glucoside, gastrodin, which is a main constituent of the Chinese herbal medicine Gastrodia elata Blume, has been widely used as an anticonvulsant, sedative, and analgesic since ancient times. However, the cellular mechanisms underlying its analgesic actions are not well understood. By utilizing a combination of behavioral surveys and electrophysiological recordings, the present study investigated the role of gastrodin in an experimental rat model of STZ-induced PDN and to further explore the underlying cellular mechanisms. Intraperitoneal administration of gastrodin effectively attenuated both the mechanical allodynia and thermal hyperalgesia induced by STZ injection. Whole-cell patch clamp recordings were obtained from nociceptive, capsaicin-sensitive small diameter neurons of the intact dorsal root ganglion (DRG). Recordings from diabetic rats revealed that the abnormal hyperexcitability of neurons was greatly abolished by application of GAS. To determine which currents were involved in the antinociceptive action of gastrodin, we examined the effects of gastrodin on transient sodium currents (I NaT) and potassium currents in diabetic small DRG neurons. Diabetes caused a prominent enhancement of I NaT and a decrease of potassium currents, especially slowly inactivating potassium currents (I AS); these effects were completely reversed by GAS in a dose-dependent manner. Furthermore, changes in activation and inactivation kinetics of I NaT and total potassium current as well as I AS currents induced by STZ were normalized by GAS. This study provides a clear cellular basis for the peripheral analgesic action of gastrodin for the treatment of chronic pain, including PDN.

Inactivation of TNF-α ameliorates diabetic neuropathy in mice

Tumor necrosis factor (TNF)-α is a potent proinflammatory cytokine involved in the pathogenesis of diabetic neuropathy. We inactivated TNF-α to determine if it is a valid therapeutic target for the treatment of diabetic neuropathy. We effected the inactivation in diabetic neuropathy using two approaches: by genetic inactivation of TNF-α (TNF-α(-/-) mice) or by neutralization of TNF-α protein using the monoclonal antibody infliximab. We induced diabetes using streptozotocin in wild-type and TNF-α(-/-) mice. We measured serum TNF-α concentration and the level of TNF-α mRNA in the dorsal root ganglion (DRG) and evaluated nerve function by a combination of motor (MNCV) and sensory (SNCV) nerve conduction velocities and tail flick test, as well as cytological analysis of intraepidermal nerve fiber density (IENFD) and immunostaining of DRG for NF-κB p65 serine-276 phosphorylated and cleaved caspase-3. Compared with nondiabetic mice, TNF-α(+/+) diabetic mice displayed significant impairments of MNCV, SNCV, tail flick test, and IENFD as well as increased expression of NF-κB p65 and cleaved caspase-3 in their DRG. In contrast, although nondiabetic TNF-α(-/-) mice showed mild abnormalities of IENFD under basal conditions, diabetic TNF-α(-/-) mice showed no evidence of abnormal nerve function tests compared with nondiabetic mice. A single injection of infliximab in diabetic TNF-α(+/+) mice led to suppression of the increased serum TNF-α and amelioration of the electrophysiological and biochemical deficits for at least 4 wk. Moreover, the increased TNF-α mRNA expression in diabetic DRG was also attenuated by infliximab, suggesting infliximab's effects may involve the local suppression of TNF-α. Infliximab, an agent currently in clinical use, is effective in targeting TNF-α action and expression and amelioration of diabetic neuropathy in mice.

Early expression of injury-induced neuropeptide Y in primary sensory neurons and the cuneate nucleus in diabetic rats with median nerve transection

In this study we examined the temporal changes in neuropeptide Y (NPY) expression in dorsal root ganglion (DRG) neurons and cuneate nucleus (CN) in streptozotocin (STZ)-induced diabetic rats with or without median nerve transection (MNT). Numerous NPY-like immunoreactive (NPY-LI) neurons and fibers were detected in the DRG and CN of the diabetic MNT (DMNT) rats respectively, but not in those with diabetes-alone. Following MNT, the time-course of NPY expression pattern in the diabetic DRG and CN was similar and both peaked at 2 weeks, which was earlier than those in the non-diabetic MNT rats. Consequently, the expression of neurotrophin-3 (NT-3) immunoreactivity in DRG neurons was coincidentally decreased and reached the nadir at 2 weeks in the diabetic MNT rats, which was also earlier than that in the non-diabetic MNT rats. Following electrical stimulation of the injured nerves, the number of NPY-LI fibers became attenuated and the induced c-Fos-LI cells concurrently appeared in the ipsilateral CN. In the diabetic CN, the number of c-Fos-LI cells also peaked at 2 weeks after MNT, which was consistent with the temporal pattern of changes in NPY expression. The results suggest that in diabetes, MNT induced NPY expression via the reduction of NT-3, and electrical stimulation of the injured median nerve evoked the release of NPY and accordingly more c-Fos-LI cells were identified in the CN. Furthermore, this study demonstrated early NPY and c-Fos expression in the diabetic rats after MNT, suggesting that the development of neuropathic signs may be advanced in hyperglycemic rats.

Identification of differentially expressed genes in dorsal root ganglion in early diabetic rats

To screen and identify differentially expressed genes in the dorsal root ganglion (DRG) in early experimental diabetic rats. Diabetic model rats were induced by single intraperitoneal injection of streptozotocin (STZ). At the second week after STZ injection, the sensory nerve conduction velocities (SNCV) of sciatic nerve were measured as an indicator of neuropathy. The technique of silver-staining mRNA differential display polymerase chain reaction (DD-PCR) was used to detect the levels of differentially expressed genes in rat DRG. The cDNA fragments that displayed differentially were identified by reverse-hybridization, cloned and sequenced subsequently, and then confirmed by Northern blot. The SNCV in the diabetic model group [n = 9, (45.25+/-10.38) m/s] reduced obviously compared with the control group [n = 8, (60.10+/-11.92) m/s] (P < 0.05). Seven distinct cDNA clones, one was up-regulated gene and the others were down-regulated ones, were isolated by silver-staining mRNA differential display method and confirmed by Northern blot. According to the results of sequence alignment with GenBank data, majority of the clones had no significant sequence similarity to previously reported genes except only one that showed high homology to 6-pyruvoyl-tetrahydropterin synthase mRNA (accession No. BC059140), which had not been reported to relate to diabetic neuropathy. These differentially expressed genes in the diabetic DRG may contribute to the pathogenesis of diabetic peripheral neuropathy.

The TRPV1 receptor is associated with preferential stress in large dorsal root ganglion neurons in early diabetic sensory neuropathy

Chronic diabetic neuropathy is associated with peripheral demyelination and degeneration of nerve fibers. The mechanism(s) underlying neuronal injury in diabetic sensory neuropathy remain poorly understood. Recently, we reported increased expression and function of transient receptor potential vanilloid 1 (TRPV1) in large dorsal root ganglion (DRG) neurons in diabetic sensory neuropathy. In this study, we examined the effects of TRPV1 activation on cell injury pathways in this subpopulation of neurons in the streptozotocin-induced diabetic rat model. Large DRG neurons from diabetic (6-8 weeks) rats displayed increased oxidative stress and activation of cell injury markers compared with healthy controls. Capsaicin (CAP) treatment induced decreased labeling of MitoTracker Red and increased cytosolic cytochrome c and activation of caspase 3 in large neurons isolated from diabetic rats. CAP treatment also induced oxidative stress in large diabetic DRG neurons, which was blocked by pre-treatment with caspase or calpain inhibitor. In addition, both mu-calpain expression and calpain activity were significantly increased in DRG neurons from diabetic rats after CAP treatment. Treatment with capsazepine, a competitive TRPV1 antagonist, markedly reduced these abnormalities in vitro and prevented activation of cell injury in large DRG neurons in diabetic rats in vivo. These results suggest that activation of the TRPV1 receptor activates pathways associated with caspase-dependent and calpain-dependent stress in large DRG neurons in STZ-diabetic rats. Activation of the TRPV1 receptor may contribute to preferential neuronal stress in large DRG neurons relatively early in diabetic sensory neuropathy.

Effect of protein kinase C?? inhibitor on Ca2+ homeostasis in diabetic sensory neurons

To elucidate the direct effect of selective protein kinase Cbeta inhibitor LY333531 on diabetic sensory neurons, we examined intracellular Ca(2+) concentration in isolated rat dorsal root ganglion neurons using the fluorescent Ca(2+) indicator fura-2. The duration of calcium transients induced by high (50 mM) extracellular K in small diabetic dorsal root ganglion neurons was significantly prolonged compared with that in control neurons. This prolonged intracellular Ca concentration elevation in diabetic neurons was normalized rapidly and reversibly by LY333531 in a dose-dependent manner, and the effect of LY333531 was completely abolished by pretreating the neurons with mitochondrial calcium uniporter inhibitor, Ruthenium 360. These results suggest that LY333531 has an ameliorating effect on calcium homeostasis of diabetic sensory neurons via mitochondrial calcium buffering.

Impaired inhibitory G‐protein function contributes to increased calcium currents in rats with diabetic neuropathy

There is a growing body of evidence that sensory neuropathy in diabetes is associated with abnormal calcium signaling in dorsal root ganglion (DRG) neurons. Enhanced influx of calcium via multiple high‐threshold calcium currents is present in sensory neurons of several models of diabetes mellitus, including the spontaneously diabetic BioBred/Worchester (BB/W) rat and the chemical streptozotocin (STZ)‐induced rat. We believe that abnormal calcium signaling in diabetes has pathologic significance as elevation of calcium influx and cytosolic calcium release has been implicated in other neurodegenerative conditions characterized by neuronal dysfunction and death. Using electrophysiologic and pharmacologic techniques, the present study provides evidence that significant impairment of G‐protein‐coupled modulation of calcium channel function may underlie the enhanced calcium entry in diabetes. N‐ and P‐type voltage‐activated, high‐threshold calcium channels in DRGs are coupled to mu opiate receptors via inhibitory G(o)‐type G proteins. The responsiveness of this receptor coupled model was tested in dorsal root ganglion (DRG) neurons from spontaneously‐diabetic BB/W rats, and streptozotocin‐induced (STZ) diabetic rats. Intracellular dialysis with GTPgammaS decreased calcium current amplitude in diabetic BB/W DRG neurons compared with those of age‐matched, nondiabetic controls, suggesting that inhibitory G‐protein activity was diminished in diabetes, resulting in larger calcium currents. Facilitation of calcium current density (I(DCa)) by large‐amplitude depolarizing prepulses (proposed to transiently inactivate G proteins), was significantly less effective in neurons from BB/W and STZ‐induced diabetic DRGs. Facilitation was enhanced by intracellular dialysis with GTPgammaS, decreased by pertussis toxin, and abolished by GDPbetaS within 5 min. Direct measurement of GTPase activity using opiate‐mediated GTPgamma[(35)S] binding, confirmed that G‐protein activity was significantly diminished in STZ‐induced diabetic neurons compared with age‐matched nondiabetic controls. Diabetes did not alter the level of expression of mu opiate receptors and G‐protein alpha subunits. These studies indicate that impaired regulation of calcium channels by G proteins is an important mechanism contributing to enhanced calcium influx in diabetes.

Oxidative stress and programmed cell death in diabetic neuropathy.

Recent evidence in both animal models and human sural nerve biopsies indicates an association with oxidative stress, mitochondrial (Mt) membrane depolarization (MMD), and induction of programmed cell death (PCD). In streptozotocin (STZ)-treated diabetic rats, hyperglycemia induces typical apoptotic changes as well as swelling and disruption of the Mt cristae in diabetic dorsal root ganglion neurons (DRG) and Schwann cells (SC), but these changes are only rarely observed in control neurons. In human sural nerve biopsies, from patients with diabetic sensory neuropathy, there is transmission electromicrograph evidence of swelling and disruption of the Mt and cristae compared to patients without peripheral neuropathy. In human SH-SY5Y neurons, rat sensory neurons, and SC, in vivo, there is an increase in reactive oxygen species (ROS) after exposure to 20 mM added glucose. In parallel, there is an initial Mt membrane hyperpolarization followed by depolarization (MMD). In turn, MMD is coupled with cleavage of caspases. Various strategies aimed at inhibiting the oxidative burst, or stabilizing the DeltaPsi(M), block induction of PCD. First, growth factors such as NGF can block induction of ROS and/or stabilize the DeltaPsi(M). This, in turn, is associated with inhibition of PCD. Second, reduction of ROS generation in neuronal Mt prevents neuronal PCD. Third, up-regulation of uncoupling proteins (UCPs), which stabilize the DeltaPsi(M), blocks induction of caspase cleavage. Collectively, these findings indicate that hyperglycemic conditions observed in diabetes mellitus are associated with oxidative stress-induced neuronal and SC death, and targeted therapies aimed at regulating ROS may prove effective in therapy of diabetic neuropathy.

Morphometry of dorsal root ganglion in chronic experimental diabetic neuropathy.

Chronic hyperglycemia results in a predominantly sensory neuropathy. Recent studies suggest that dorsal root ganglion (DRG) neurons comprise a specific target and may be responsible for the important complication of diabetic sensory neuropathy, since hyperglycemia for longer than 6 months results in a vacuolar ganglionopathy with associated radiculopathy and distal sensory neuropathy. We undertook morphometric analysis of L5 DRG neurons in seven diabetic rats and six age- and sex-matched littermates. Nerve conduction studies were also performed, and neuropathy was confirmed. Diabetes was induced with streptozotocin; duration of diabetes was 12 months. The DRG count for control rats was 15,304 +/- 991 neurons. Two of seven diabetic DRG counts were reduced, but the group mean count at 14,847 plus minus 1,524 was not significantly reduced. The number of small neurons (type B) considerably exceeded that of large neurons (type A), at a ratio of 71:29. The percentage of large cells was significantly reduced in diabetic compared with control rats (P = 0.01). The large-diameter population can be subdivided into two groups; with this subdivision, the number of neurons < 50 microm was not reduced in samples from diabetic rats, but the neurons of largest size (> or = 50 microm) were significantly reduced (by 41%).