High-mobility group box 1 contributes to mechanical allodynia and spinal astrocytic activation in a mouse model of type 2 diabetes

Chronic pain is one of the most common complications of diabetes. However, current treatments for diabetic pain are usually unrealistic because the underlying mechanisms are far from being clear. Immerging studies have implicated immune factors as key players in the diabetic pain. High-mobility group box 1 (HMGB1) is an important mediator of inflammatory response, but its role in diabetic pain is unclear. In the present study, we observed that db/db mice (a model of type 2 diabetes) developed persistent mechanical allodynia from postnatal 2 months. Western blot showed that in postnatal 2-5 months, HMGB1 was significantly higher than that of the heterozygous littermates (db/+) mice. Intrathecal injection of a HMGB1 neutralizing antibody (anti-HMGB1) inhibited mechanical allodynia. Immunostaining data showed that compared with db/+ and C57 mice (postnatal 4 months), glial fibrillary acidic protein (GFAP) staining was significantly increased in the spinal cord of db/db mice. Anti-HMGB1 could effectively decrease GFAP expression. Real-time PCR showed that in postnatal 4 months, db/db mice induced significant increases of TNF-alpha, IL-1β, IL-6 and monocyte chemoattractant protein-1 (MCP-1) in the spinal dorsal horn, while anti-HMGB1 (50 μg) effectively inhibited the up-regulation of these inflammatory mediators. Our results indicate that HMGB1 is significantly up-regulated in the spinal cord of type 2 diabetes, and inhibiting HMGB1 may provide a novel treatment for diabetic pain.     

Comparative study of the distribution of the alpha-subunits of voltage-gated sodium channels in normal and axotomized rat dorsal root ganglion neurons

We compared the distribution of the α‐subunit mRNAs of voltage‐gated sodium channels Nav1.1–1.3 and Nav1.6–1.9 and a related channel, Nax, in histochemically identified neuronal subpopulations of the rat dorsal root ganglia (DRG). In the naïve DRG, the expression of Nav1.1 and Nav1.6 was restricted to A‐fiber neurons, and they were preferentially expressed by TrkC neurons, suggesting that proprioceptive neurons possess these channels. Nav1.7, ‐1.8, and ‐1.9 mRNAs were more abundant in C‐fiber neurons compared with A‐fiber ones. Nax was evenly expressed in both populations. Although Nav1.8 and ‐1.9 were preferentially expressed by TrkA neurons, other α‐subunits were expressed independently of TrkA expression. Actually, all IB4⁺ neurons expressed both Nav1.8 and ‐1.9, and relatively limited subpopulations of IB4⁺ neurons (3% and 12%, respectively) expressed Nav1.1 and/or Nav1.6. These findings provide useful information in interpreting the electrophysiological characteristics of some neuronal subpopulations of naïve DRG. After L5 spinal nerve ligation, Nav1.3 mRNA was up‐regulated mainly in A‐fiber neurons in the ipsilateral L5 DRG. Although previous studies demonstrated that nerve growth factor (NGF) and glial cell‐derived neurotrophic factor (GDNF) reversed this up‐regulation, the Nav1.3 induction was independent of either TrkA or GFRα1 expression, suggesting that the induction of Nav1.3 may be one of the common responses of axotomized DRG neurons without a direct relationship to NGF/GDNF supply. J. Comp. Neurol. 510:188–206, 2008. © 2008 Wiley‐Liss, Inc.     

Contactin regulates the current density and axonal expression of tetrodotoxin-resistant but not tetrodotoxin-sensitive sodium channels in DRG neurons

Contactin, a glycosyl-phosphatidylinositol (GPI)-anchored predominantly neuronal cell surface glycoprotein, associates with sodium channels Nav1.2, Nav1.3 and Nav1.9, and enhances the density of these channels on the plasma membrane in mammalian expression systems. However, a detailed functional analysis of these interactions and of untested putative interactions with other sodium channel isoforms in mammalian neuronal cells has not been carried out. We examined the expression and function of sodium channels in small-diameter dorsal root ganglion (DRG) neurons from contactin-deficient (CNTN-/-) mice, compared to CNTN+/+ litter mates. Nav1.9 is preferentially expressed in isolectin B4 (IB4)-positive neurons and thus we used this marker to subdivide small-diameter DRG neurons. Using whole-cell patch-clamp recording, we observed a greater than two-fold reduction of tetrodotoxin-resistant (TTX-R) Nav1.8 and Nav1.9 current densities in IB4+ DRG neurons cultured from CNTN-/- vs. CNTN+/+ mice. Current densities for TTX-sensitive (TTX-S) sodium channels were unaffected. Contactin's effect was selective for IB4+ neurons as current densities for both TTX-R and TTX-S channels were not significantly different in IB4- DRG neurons from the two genotypes. Consistent with these results, we have demonstrated a reduction in Nav1.8 and Nav1.9 immunostaining on peripherin-positive unmyelinated axons in sciatic nerves from CNTN-/- mice but detected no changes in the expression for the two major TTX-S channels Nav1.6 and Nav1.7. These data provide evidence of a role for contactin in selectively regulating the cell surface expression and current densities of TTX-R but not TTX-S Na+ channel isoforms in nociceptive DRG neurons; this regulation could modulate the membrane properties and excitability of these neurons.     

Morphometric analysis of obesity (ob/ob)- and diabetes (db/db)-associated hypothalamic neuronal degeneration in C57BL/KsJ mice

The influence of the obese (ob/ob) and diabetes (db/db) genetic mutations on hypothalamic structure was investigated in C57BL/KsJ and C57BL/6J mice strains by morphometric analysis of medial basal nuclei which are recognized to possess glucoregulatory neurons. Brains were collected and prepared for histomorphometric analysis at selected times following the development of expressed obesity and diabetes (Type II, non-insulin dependent) syndromes in order to compare both the strain and genomic influences on neuronal viability in the hypothalamic ventromedial (VMH) and arcuate (ARC) nuclei of mutant and age-matched control mice. The severity of each syndrome was determined by monitoring the concomitant changes in body weight and blood glucose levels in all groups. Both (db/db) and (ob/ob) mutant C57BL/KsJ mice exhibited an increase in the number and distribution of degenerated neurons in the VMH and ARC nuclei relative to corresponding controls. The mutation-associated exacerbation of the normal age-related neuronal loss, as observed in control MBH nuclei, was temporally associated with the overt expression of the hyperglycemic component of the obese and diabetes syndromes in aging C57BL/KsJ mice. No temporal or causal relationships were noted between the enhanced rate of premature neuronal degeneration, and either body weight or blood glucose levels, in either (db/db) or (ob/ob) C57BL/6J mice relative to controls. These data suggest that the hyperglycemic condition which characterizes the (ob/ob) and (db/db) mutant C57BL/KsJ mice is causally associated with the pronounced, premature MBH neuronal degeneration in these mouse strains. Neuronal changes were not pronounced when the genetic mutations were expressed in C57BL/6J mice. The accompanying alterations in brain glucose metabolism, hormone sensitivity, bioamine content and function which are recognized to occur in these mutant C57BL/KsJ mice may be causally associated consequences of the observed changes in MBH structural integrity and neuronal competence, with the severity of the mutation-associated changes being related to genetic background of the murine strain.     

Altered sodium currents in auditory neurons of congenitally deaf mice

Sodium currents are essential for action potential generation and propagation in most excitable cells. Appropriate tuning of these currents can be modulated both developmentally and in response to activity. Here we use a mouse model of congenital deafness (dn/dn- asymptomatic deafness associated with hair cell degeneration) to investigate the effect of lack of activity in the expression of Na(+) currents in neurons from the medial nucleus of the trapezoid body (MNTB). Patch-clamp recordings show that at postnatal day (P) 14, both normal and deaf mice display a significant amount of persistent and resurgent Na(+) currents. However, the persistent current is greater in deaf mice than in normal mice, and resurgent current kinetics are slower in deaf mice. At P7, resurgent currents are not present in either group. MNTB immunohistochemistry demonstrates that Nav1.1 subunits are expressed postsynaptically in both P14 normal and deaf mice, while postsynaptic Nav1.6 staining was only observed in deaf mice. Labelling of Nav1.6 subunits in different age groups revealed that at younger ages (P7), both normal and deaf mice express this protein. Nav1.6 staining was not observed in MNTB neurons of P28 normal mice, whereas it is maintained in deaf mice cells until much later (P28). At P7, none of the groups displayed resurgent currents (despite the detection of Nav1.6 subunits at this age group); this suggests that factors other than alpha subunits are important for modulating these currents in MNTB cells. Our results emphasize the importance of activity during development in regulating Na(+) channels.     

Role of hippocampal sodium channel Nav1.6 in kindling epileptogenesis

Purpose:   Central nervous system plasticity is essential for normal function, but can also reinforce abnormal network behavior, leading to epilepsy and other disorders. The role of altered ion channel expression in abnormal plasticity has not been thoroughly investigated. Nav1.6 is the most abundantly expressed sodium channel in the nervous system. Because of its distribution in the cell body and axon initial segment, Nav1.6 is crucial for action potential generation. The goal of the present study was to investigate the possible role of changes in Nav1.6 expression in abnormal, activity-dependent plasticity of hippocampal circuits.
Methods:   We studied kindling, a form of abnormal activity-dependent facilitation. We investigated: (1) sodium channel protein expression by immunocytochemistry and sodium channel messenger RNA (mRNA) by in situ hybridization, (2) sodium current by patch clamp recordings, and (3) rate of kindling by analysis of seizure behavior. The initiation, development, and expression of kindling in wild-type mice were compared to Nav1.6 +/− med ^tg mice, which have reduced expression of Nav1.6.
Results:   We found that kindling was associated with increased expression of Nav1.6 protein and mRNA, which occurred selectively in hippocampal CA3 neurons. Hippocampal CA3 neurons also showed increased persistent sodium current in kindled animals compared to sham-kindled controls. Conversely, Nav1.6 +/− med ^tg mice resisted the initiation and development of kindling.
Discussion:   These findings suggest an important mechanism for enhanced excitability, in which Nav1.6 may participate in a self-reinforcing cycle of activity-dependent facilitation in the hippocampus. This mechanism could contribute to both normal hippocampal function and to epilepsy and other common nervous system disorders.     

SOD2 protects neurons from injury in cell culture and animal models of diabetic neuropathy

Hyperglycemia-induced oxidative stress is an inciting event in the development of diabetic complications including diabetic neuropathy. Our observations of significant oxidative stress and morphological abnormalities in mitochondria led us to examine manganese superoxide dismutase (SOD2), the enzyme responsible for mitochondrial detoxification of oxygen radicals. We demonstrate that overexpression of SOD2 decreases superoxide (O(2)(-)) in cultured primary dorsal root ganglion (DRG) neurons and subsequently blocks caspase-3 activation and cellular injury. Underexpression of SOD2 in dissociated DRG cultures from adult SOD2(+/-) mice results in increased levels of O2-, activation of caspase-3 cleavage and decreased neurite outgrowth under basal conditions that are exacerbated by hyperglycemia. These profound changes in sensory neurons led us to explore the effects of decreased SOD2 on the development of diabetic neuropathy (DN) in mice. DN was assessed in SOD2(+/-) C57BL/6J mice and their SOD2(+/+) littermates following streptozotocin (STZ) treatment. These animals, while hyperglycemic, do not display any signs of DN. DN was observed in the C57BL/6Jdb/db mouse, and decreased expression of SOD2 in these animals increased DN. Our data suggest that SOD2 activity is an important cellular modifier of neuronal oxidative defense against hyperglycemic injury.     

Morphometric analysis of medial basal hypothalamic neuronal degeneration in diabetes (db/db) mutant C57BL/KsJ mice: relation to age and hyperglycemia

Age- and diabetes-related neuronal degenerative changes were morphometrically evaluated in the arcuate (ARC) and ventromedial (VMH) hypothalamic nuclei of control (+/?) and diabetic (db/db) C57BL/KsJ mice between 4 and 16 weeks of age. By 4 weeks of age, (db/db) mice exhibited marked obesity and hyperglycemia relative to controls. An increase in the population of degenerated ARC neurons was detected at 8 weeks of age in (db/db) mice relative to (+/?) animals. By 16 weeks of age, a significant increase in the number of degenerated VMH neurons in (db/db) mice was found, relative to controls. In addition, the neuronal density (neurons/mm2 area of nucleus) of both the ARC and VMH nuclei was found to be depressed in (db/db) mice, relative to controls, by 16 weeks of age. These data suggest that the normal degenerative loss of ARC and VMH neurons that occurs with age in normal mice is enhanced in the (db/db) mouse. These findings suggest that a functional alteration in hypothalamic nuclei which are recognized to modulate autonomic, pancreatic and pituitary activity may be associated with the onset or expression of the diabetic condition in the C57BL/KsJ (db/db) mouse.     

Expression and localization of the Nav1.9 sodium channel in enteric neurons and in trigeminal sensory endings: implication for intestinal reflex function and orofacial pain

The Nav1.9 sodium channel is expressed in nociceptive DRG neurons where it contributes to spontaneous pain behavior after peripheral inflammation. Here, we used a newly developed antibody to investigate the distribution of Nav1.9 in rat and mouse trigeminal ganglion (TG) nerve endings and in enteric nervous system (ENS). In TGs, Nav1.9 was expressed in the soma of small- and medium-sized, peripherin-positive neurons. Nav1.9 was present along trigeminal afferent fibers and at terminals in lip skin and dental pulp. In the ENS, Nav1.9 was detected within the soma and proximal axons of sensory, Dogiel type II, myenteric and submucosal neurons. Immunological data were correlated with the detection of persistent TTX-resistant Na(+) currents sharing similar properties in DRG, TG and myenteric neurons. Collectively, our data support a potential role of Nav1.9 in the transmission of trigeminal pain and the regulation of intestinal reflexes. Nav1.9 might therefore constitute a molecular target for therapeutic treatments of orofacial pain and gastrointestinal syndromes.     

Diabetes-induced reduction of neuronal survival in hypotonic environments in culture

Dorsal root ganglion (DRG) neurons from streptozotocin (STZ)-induced diabetic and normal C57BL mice were exposed to three different hypotonic environments ( , , and 1/8 osmolar solutions). After rapid applications of these hypotonic solutions to the neurons, the cell volume autoregulatory mechanism operated in osmolar solution but was disrupted in superhypotonic solutions below osmolar in both kinds of mice. None of the neurons could survive 12 h after treatment with superhypotonic solutions. On the other hand, a gradual reduction of osmolarity of the culture medium enabled neurons in the normal mice to survive in and osmolar solutions as well as i an isotonic solution. However, this reduction of osmolarity increased neuronal cell death in the diabetic mice. These results suggest that the ability of DRG neurons to survive in hypotonic environments in culture may be lost in diabetes.     

Dysregulation of sodium channel expression in cortical neurons in a rodent model of absence epilepsy

Due to the involvement of cortical neurons in spike-wave discharge (SWD) initiation, and the contribution of voltage-gated sodium channels (VGSCs) to neuronal firing, we examined alterations in the expression of VGSC mRNA and protein in cortical neurons in the WAG/Rij absence epileptic rat. WAG/Rij rats were compared to age-matched Wistar control rats at 2, 4, and 6 months. Continuous EEG data was recorded, and percent time in SWD was determined. Tissue from different cortical locations from WAG/Rij and Wistar rats was analyzed for VGSC mRNA (by quantitative PCR) and protein (by immunocytochemistry). SWDs increased with age in WAG/Rij rats. mRNA levels for sodium channels Nav1.1 and Nav1.6, but not Nav1.2, were found to be up-regulated selectively within the facial somatosensory cortex (at AP +0.0, ML +6.0 mm). Protein levels for Nav1.1 and Nav1.6 were up-regulated in layer II–IV cortical neurons in this region of cortex. No significant changes were seen in adjacent regions or other brain areas, including the pre-frontal and occipital cortex. In the WAG/Rij model of absence epilepsy, we identified a specific region of cortex, in layer II–IV neurons on the lateral convexity of the cortex in the facial somatosensory area, where mRNA and protein expression of sodium channel genes Nav1.1 and Nav1.6 are up-regulated. This region of cortex approximately matches the electrophysiologically determined region of seizure onset. Changes in the expression of Nav1.1 and Nav1.6 parallel age-dependent increases in seizure frequency and duration.     

Downregulation of P2X3 receptor-dependent sensory functions in A/J inbred mouse strain

There is large variability in the various pain responses including those to tissue injury among inbred mouse strains. However, the determinant factors for the strain-specific differences remain unknown. The P2X3 sensory-specific ATP-gated channel has been implicated as a damage-sensing molecule that evokes a pain sensation by receiving endogenous ATP from injured tissue. In this study, to clarify the contribution of the sensory P2X3 signalling to strain-specific differences in tissue injury pain, we examined whether the P2X3-mediated in vivo and in vitro responses in dorsal root ganglion (DRG) neurons are changed in the A/J inbred mouse strain, which is known to be resistant to tissue injury pain caused by formalin. Here we found that A/J mice exhibited a low magnitude of nocifensive behaviour induced by the P2X agonist alpha,beta-methylene ATP (alpha beta meATP) into the hindpaw compared with C57BL/6 J mice. This behaviour was blocked by P2X3 antisense oligodeoxynucleotides. The low magnitude of the in vivo pain sensation could be observed similarly in the in vitro response; the increase in the intracellular Ca(2+) increase by alpha beta meATP in capsaicin-sensitive DRG neurons from A/J mice was significantly lower than that from C57BL/6 J mice. In A/J DRG neurons the P2X3 protein level was significantly lower compared with C57BL/6 J DRG neurons. The change in P2X3 protein was selective because P2X2 protein was expressed equally in both strains. The present study suggests that the downregulation of sensory P2X3 could be one of the molecular predispositions to low sensitivity to tissue injury pain in the A/J inbred mouse strain.     

Changes in P2X receptor responses of sensory neurons from P2X3-deficient mice

Dorsal root ganglion (DRG) neurons respond to ATP with transient, persistent or biphasic inward currents. In contrast, the ATP responses in nodose neurons are persistent. These sustained currents are also heterogeneous, with one component being accounted for by P2X2/3 receptors, and the residual response probably mediated by P2X2 receptors, although the direct evidence for this has been lacking. In the present study, we examined the P2X receptors on DRG and nodose neurons from P2X3-deficient (P2X3-/-) mice, using whole cell voltage-clamp recording and immunohistochemistry. We found that all P2X3-/- DRG neurons lacked rapidly desensitizing response to ATP, and both DRG and nodose neurons from P2X3-null mutant mice no longer responded to alpha,beta-methylene ATP (alphabetameATP). In contrast, ATP evoked persistent inward current in 12% of DRG neurons and 84% of nodose neurons from P2X3-/- mice. This retained persistent response to ATP on nodose neurons had an EC50 for ATP of 77 microm, was antagonized by Cibacron blue and pyridoxal-5-phosphate-6-azophenyl-2',4'-disulphonic acid, potentiated by Zn2+ and acidification, but not enhanced by ivermectin or diinosine pentaphosphate. 2',3'-O-Trinitrophenyl-ATP antagonized this response with an IC50 of 8 microm. All these properties are consistent with those of recombinant P2X2 homomeric receptors. Furthermore, specific P2X2 receptor immunoreactivity detected in wild-type sensory neurons was unaltered in null mutant mice. Therefore, the alphabetameATP-insensitive persistent responses on nodose neurons are likely to be mediated by P2X2 homomers, which contribute to 60% of currents evoked by 100 microm ATP in the wild type.     

Growth Differentiation Factor-15 Produces Analgesia by Inhibiting Tetrodotoxin-Resistant Nav1.8 Sodium Channel Activity in Rat Primary Sensory Neurons

Growth differentiation factor 15 (GDF-15) is a member of the transforming growth factor-β superfamily. It is widely distributed in the central and peripheral nervous systems. Whether and how GDF-15 modulates nociceptive signaling remains unclear. Behaviorally, we found that peripheral GDF-15 significantly elevated nociceptive response thresholds to mechanical and thermal stimuli in naïve and arthritic rats. Electrophysiologically, we demonstrated that GDF-15 decreased the excitability of small-diameter dorsal root ganglia (DRG) neurons. Furthermore, GDF-15 concentration-dependently suppressed tetrodotoxin-resistant sodium channel Nav1.8 currents, and shifted the steady-state inactivation curves of Nav1.8 in a hyperpolarizing direction. GDF-15 also reduced window currents and slowed down the recovery rate of Nav1.8 channels, suggesting that GDF-15 accelerated inactivation and slowed recovery of the channel. Immunohistochemistry results showed that activin receptor-like kinase-2 (ALK2) was widely expressed in DRG medium- and small-diameter neurons, and some of them were Nav1.8-positive. Blockade of ALK2 prevented the GDF-15-induced inhibition of Nav1.8 currents and nociceptive behaviors. Inhibition of PKA and ERK, but not PKC, blocked the inhibitory effect of GDF-15 on Nav1.8 currents. These results suggest a functional link between GDF-15 and Nav1.8 in DRG neurons via ALK2 receptors and PKA associated with MEK/ERK, which mediate the peripheral analgesia of GDF-15.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12264-021-00709-5.     

TNF-α enhances the currents of voltage gated sodium channels in uninjured dorsal root ganglion neurons following motor nerve injury

The ectopic discharges observed in uninjured dorsal root ganglion (DRG) neurons following various lesions of spinal nerves have been attributed to functional alterations of voltage-gated sodium channels (VGSCs). Such mechanisms may be important for the development of neuropathic pain. However, the pathophysiology underlying the functional modulation of VGSCs following nerve injury is largely unknown. Here, we studied this issue with use of a selective lumbar 5 ventral root transection (L5-VRT) model, in which dorsal root ganglion (DRG) neurons remain intact. We found that the L5-VRT increased the current densities of TTX-sensitive Na channels as well as currents in Nav1.8, but not Nav1.9 channels in uninjured DRG neurons. The thresholds of action potentials decreased and firing rates increased in DRG neurons following L5-VRT. As we found that levels of tumor necrosis factor-alpha (TNF-α) increased in cerebrospinal fluid (CSF) and in DRG tissue after L5-VRT, we tested whether the increased TNF-α might result in the changes in sodium channels. Indeed, recombinant rat TNF (rrTNF) enhanced the current densities of TTX-S and Nav1.8 in cultured DRG neurons dose-dependently. Furthermore, genetic deletion of TNF receptor 1 (TNFR-1) in mice attenuated the mechanical allodynia and prevented the increase in sodium currents in DRG neurons induced by L5-VRT. These data suggest that the increase in sodium currents in uninjured DRG neurons following nerve injury might be mediated by over-production of TNF-α.     

Differential Inhibition of Nav1.7 and Neuropathic Pain by Hybridoma-Produced and Recombinant Monoclonal Antibodies that Target Nav1.7

The voltage-gated Na~+ channel subtype Nav1.7 is important for pain and itch in rodents and humans. We previously showed that a Nav1.7-targeting monoclonal antibody(SVmab) reduces Na+ currents and pain and itch responses in mice. Here, we investigated whether recombinant SVmab(rSVmab) binds to and blocks Nav1.7 similar to SVmab. ELISA tests revealed that SVmab was capable of binding to Nav1.7-expressing HEK293 cells,mouse DRG neurons, human nerve tissue, and the voltagesensor domain Ⅱ of Nav1.7. In contrast, rSVmab showed no or weak binding to Nav1.7 in these tests. Patch-clamp recordings showed that SVmab, but not rSVmab, markedly inhibited Na+ currents in Nav1.7-expressing HEK293 cells. Notably, electrical field stimulation increased the blocking activity of SVmab and rSVmab in Nav1.7-expressing HEK293 cells. SVmab was more effective than rSVmab in inhibiting paclitaxel-induced mechanical allodynia. SVmab also bound to human DRG neurons and inhibited their Na~+ currents. Finally, potential reasons for the differential efficacy of SVmab and rSVmab and future directions are discussed.