Shuangsong Hong

Sodium Channel β2 Subunits Regulate Tetrodotoxin-Sensitive Sodium Channels in Small Dorsal Root Ganglion Neurons and Modulate the Response to Pain

Voltage-gated sodium channel (Nav1) β2 subunits modulate channel gating, assembly, and cell-surface expression in CNS neurons in vitro and in vivo. β2 expression increases in sensory neurons after nerve injury, and development of mechanical allodynia in the spared nerve injury model is attenuated in β2-null mice. Thus, we hypothesized that β2 modulates electrical excitability in dorsal root ganglion (DRG) neurons in vivo. We compared sodium currents (INa) in small DRG neurons from β2+/+ and β2−/− mice to determine the effects of β2 on tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) Nav1 in vivo. Small-fast DRG neurons acutely isolated from β2−/− mice showed significant decreases in TTX-S INa compared with β2+/+ neurons. This decrease included a 51% reduction in maximal sodium conductance with no detectable changes in the voltage dependence of activation or inactivation. TTX-S, but not TTX-R, INa activation and inactivation kinetics in these cells were slower in β2−/− mice compared with controls. The selective regulation of TTX-S INa was supported by reductions in transcript and protein levels of TTX-S Nav1s, particularly Nav1.7. Low-threshold mechanical sensitivity was preserved in β2−/− mice, but they were more sensitive to noxious thermal stimuli than wild type whereas their response during the late phase of the formalin test was attenuated. Our results suggest that β2 modulates TTX-S Nav1 mRNA and protein expression resulting in increased TTX-S INa and increases the rates of TTX-S Nav1 activation and inactivation in small-fast DRG neurons in vivo. TTX-R INa were not significantly modulated by β2.

Reciprocal Changes in Vanilloid (TRPV1) and Endocannabinoid (CB1) Receptors Contribute to Visceral Hyperalgesia in the Water Avoidance Stressed Rat

Background: Increasing evidence suggests that chronic stress plays an important role in the pathophysiology of several functional gastrointestinal disorders. We investigated whether cannabinoid receptor 1 (CB1) and vanilloid receptor 1 (TRPV1; transient receptor potential vanilloid 1) are involved in stress-induced visceral hyperalgesia. Methods: Male rats were exposed to 1 h water avoidance (WA) stress daily for 10 consecutive days. The visceromotor response (VMR) to colorectal distension (CRD) was measured. Immunofluorescence and western blot analysis were used to assess the expression of CB1 and TRPV1 receptors in dorsal root ganglion (DRG) neurons. Results: WA stressed rats demonstrated a significant increase in the serum corticosterone levels and faecal pellet output compared to controls supporting stimulation of the hypothalamic–pituitary–adrenal (HPA) axis. The VMR increased significantly at pressures of 40 and 60 mm Hg in WA stress rats compared with controls, respectively, and was associated with hyperalgesia. The endogenous CB1 agonist anandamide was increased significantly in DRGs from stressed rats. Immunofluorescence and western blot analysis showed a significant decrease in CB1 and a reciprocal increase in TRPV1 expression and phosphorylation in DRG neurons from stressed rats. These reciprocal changes in CB1 and TRPV1 were reproduced by treatment of control DRGs with anandamide in vitro. In contrast, treatment of control DRGs in vitro with the CB1 receptor agonist WIN 55,212-2 decreased the levels of TRPV1 and TRPV1 phosphorylation. Treatment of WA stress rats in situ with WIN 55,212-2 or the TRPV1 antagonist capsazepine prevented the development of visceral hyperalgesia and blocked the upregulation of TRPV1. Conclusions: These results suggest that the endocannabinoid (CB1) and TRP (TRPV1) pathways may play a potentially important role in stress-induced visceral hyperalgesia.

Corticosterone Mediates Reciprocal Changes in CB 1 and TRPV1 Receptors in Primary Sensory Neurons in the Chronically Stressed Rat

BACKGROUND & AIMS Chronic stress is associated with visceral hyperalgesia in functional gastrointestinal disorders. We investigated whether corticosterone plays a role in chronic psychological stress-induced visceral hyperalgesia. METHODS Male rats were subjected to 1-hour water avoidance (WA) stress or subcutaneous corticosterone injection daily for 10 consecutive days in the presence or absence of corticoid-receptor antagonist RU-486 and cannabinoid-receptor agonist WIN55,212-2. The visceromotor response to colorectal distension was measured. Receptor protein levels were measured and whole-cell patch-clamp recordings were used to assess transient receptor potential vanilloid type 1 (TRPV1) currents in L6-S2 dorsal root ganglion (DRG) neurons. Mass spectrometry was used to measure endocannabinoid anandamide content. RESULTS Chronic WA stress was associated with visceral hyperalgesia in response to colorectal distension, increased stool output and reciprocal changes in cannabinoid receptor 1 (CB1) (decreased) and TRPV1 (increased) receptor expression and function. Treatment of WA stressed rats with RU-486 prevented these changes. Control rats treated with serial injections of corticosterone in situ showed a significant increase in serum corticosterone associated with visceral hyperalgesia, enhanced anandamide content, increased TRPV1, and decreased CB1 receptor protein levels, which were prevented by co-treatment with RU-486. Exposure of isolated control L6-S2 DRGs in vitro to corticosterone reproduced the changes in CB1 and TRPV1 receptors observed in situ, which was prevented by co-treatment with RU-486 or WIN55,212-2. CONCLUSIONS These results support a novel role for corticosterone to modulate CB1 and TRPV1-receptor pathways in L6-S2 DRGs in the chronic WA stressed rat, which contributes to visceral hyperalgesia observed in this model.

Diabetic autonomic neuropathy: evidence for apoptosis in situ in the rat

Abstract  We examined the hypothesis that activation of the apoptosis cascade occurs relatively early in diabetes mellitus affecting three distinct neuronal populations that are involved in regulating gut function: (i) dorsal root ganglion (DRG), (ii) vagus nodose ganglion and (iii) colon myenteric plexus. A validated streptozotocin‐induced diabetic rat model and age‐matched healthy controls were studied. After 4–8 weeks of diabetes the animals were anaesthetized, fixed in situ and the relevant tissues removed. After 1 month of diabetes some animals were treated with insulin for 2 weeks to restore euglycaemia. Apoptosis was measured using immunohistochemical detection of activated caspase‐3 and terminal deoxynucleotidyl transferase‐mediated dUTP nick‐end labelling (TUNEL)‐positive cells in adjacent sections in neurones (PGP 9.5‐positive cells). The level of apoptosis was confirmed using double‐label assessment of caspase‐3 and TUNEL in DRG preparations. Caspase‐3 immunoreactive neurones demonstrated a range in staining intensity. When all grades of staining were included, 6–8% of the DRG, nodose ganglia and myenteric neurones were immunoreactive in the preparations from diabetic rats compared with 0.2–0.5% in controls. Neurones staining positive for both caspase‐3 and TUNEL accounted for 1–2% of the total neuronal population in all three preparations in diabetic rats compared with 0.1–0.2% in controls (P < 0.05). Insulin treatment reversed the percentage of TUNEL‐positive neurones in diabetic rats to control levels. Activation of the apoptosis cascade occurs relatively early in diabetic autonomic neuropathy and may contribute to the pathophysiology of this disorder.

Sera from patients with type 2 Diabetes and Neuropathy Induce Autophagy and Colocalization with Mitochondria in SY5Y cells

The etiology of diabetic neuropathy is multifactorial and not fully elucidated, although oxidative stress and mitochondrial dysfunction are major factors. We reported previously that complement-inactivated sera from type 2 diabetic patients with neuropathy induce apoptosis in cultured neuronal cells, possibly through an autoimmune immunoglobulin-mediated pathway. Recent evidence supports an emerging role for autophagy in a variety of diseases. Here we report that exposure of human neuroblastoma SH-SY5Y cells to sera from type 2 diabetic patients with neuropathy is associated with increased levels of autophagosomes that is likely mediated by increased titers of IgM or IgG autoimmune immunoglobulins. The increased presence of macroautophagic vesicles was monitored using a specific immunohistochemical marker for autophagosomes, anti-LC3-II immunoreactivity, as well as the immunohistochemical signal for beclin-1, and was associated with increased co-localization with mitochondria in the cells exposed to diabetic neuropathic sera. We also report that dorsal root ganglia removed from streptozotocin-induced diabetic rats exhibit increased levels of autophagosomes and co-localization with mitochondria in neuronal soma, concurrent with enhanced binding of IgG and IgM autoimmune immunoglobulins. To our knowledge, this is the first evidence that the presence of autophagosomes is increased by a serum factor, likely autoantibody(ies) in a pathological condition. Stimulation of autophagy by an autoantibody-mediated pathway can provide a critical link between the immune system and the loss of function and eventual demise of neuronal tissue in type 2 diabetes.

Epigenetic Regulation of Genes That Modulate Chronic Stress-Induced Visceral Pain in the Peripheral Nervous System

BACKGROUND & AIMS Chronic stress alters the hypothalamic-pituitary-adrenal axis, increases gut motility, and increases the perception of visceral pain. We investigated whether epigenetic mechanisms regulate chronic stress-induced visceral pain in the peripheral nervous systems of rats. METHODS Male rats were subjected to 1 hour of water avoidance stress each day, or given daily subcutaneous injections of corticosterone, for 10 consecutive days. L4-L5 and L6-S2 dorsal root ganglia (DRG) were collected and compared between stressed and control rats (placed for 1 hour each day in a tank without water). Levels of cannabinoid receptor 1 (CNR1), DNA (cytosine-5-)-methyltransferase 1 (DNMT1), transient receptor potential vanilloid type 1 (TRPV1), and EP300 were knocked down in DRG neurons in situ with small interfering RNAs. We measured DNA methylation and histone acetylation at genes encoding the glucocorticoid receptor (NR3C1), CNR1, and TRPV1. Visceral pain was measured in response to colorectal distention. RESULTS Chronic stress was associated with increased methylation of the Nr3c1 promoter and reduced expression of this gene in L6-S2, but not L4-L5, DRGs. Stress also was associated with up-regulation in DNMT1-associated methylation of the Cnr1 promoter and down-regulation of glucocorticoid-receptor-mediated expression of CNR1 in L6-S2, but not L4-L5, DRGs. Concurrently, chronic stress increased expression of the histone acetyltransferase EP300 and increased histone acetylation at the Trpv1 promoter and expression of the TRPV1 receptor in L6-S2 DRG neurons. Knockdown of DNMT1 and EP300 in L6-S2 DRG neurons of rats reduced DNA methylation and histone acetylation, respectively, and prevented chronic stress-induced increases in visceral pain. CONCLUSIONS Chronic stress increases DNA methylation and histone acetylation of genes that regulate visceral pain sensation in the peripheral nervous system of rats. Blocking epigenetic regulatory pathways in specific regions of the spinal cord might be developed to treat patients with chronic abdominal pain.

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 μ‐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.

Corticosterone mediates stress-related increased intestinal permeability in a region-specific manner.

Background  Chronic psychological stress (CPS) is associated with increased intestinal epithelial permeability and visceral hyperalgesia. It is unknown whether corticosterone (CORT) plays a role in mediating alterations of epithelial permeability in response to CPS.

Chronic Stress and Peripheral Pain: Evidence for Distinct, Region-specific Changes in Visceral and Somatosensory Pain Regulatory Pathways

Chronic stress alters the hypothalamic-pituitary-adrenal (HPA) axis and enhances visceral and somatosensory pain perception. It is unresolved whether chronic stress has distinct effects on visceral and somatosensory pain regulatory pathways. Previous studies reported that stress-induced visceral hyperalgesia is associated with reciprocal alterations of endovanilloid and endocannabinoid pain pathways in DRG neurons innervating the pelvic viscera. In this study, we compared somatosensory and visceral hyperalgesia with respect to differential responses of peripheral pain regulatory pathways in a rat model of chronic, intermittent stress. We found that chronic stress induced reciprocal changes in the endocannabinoid 2-AG (increased) and endocannabinoid degradation enzymes COX-2 and FAAH (decreased), associated with down-regulation of CB1 and up-regulation of TRPV1 receptors in L6-S2 DRG but not L4-L5 DRG neurons. In contrast, sodium channels Nav1.7 and Nav1.8 were up-regulated in L4-L5 but not L6-S2 DRGs in stressed rats, which was reproduced in control DRGs treated with corticosterone in vitro. The reciprocal changes of CB1, TRPV1 and sodium channels were cell-specific and observed in the sub-population of nociceptive neurons. Behavioral assessment showed that visceral hyperalgesia persisted, whereas somatosensory hyperalgesia and enhanced expression of Nav1.7 and Nav1.8 sodium channels in L4-L5 DRGs normalized 3 days after completion of the stress phase. These data indicate that chronic stress induces visceral and somatosensory hyperalgesia that involves differential changes in endovanilloid and endocannabinoid pathways, and sodium channels in DRGs innervating the pelvic viscera and lower extremities. These results suggest that chronic stress-induced visceral and lower extremity somatosensory hyperalgesia can be treated selectively at different levels of the spinal cord.

Neurogenic chronic intestinal pseudo-obstruction: antineuronal antibody-mediated activation of autophagy via Fas.

BACKGROUND & AIMS Activation of autoimmune pathways has been implicated as a contributing mechanism to the pathophysiology in some patients with chronic intestinal pseudoobstruction (CIP). In this study we tested the hypothesis that sera from a subpopulation of patients with CIP contain autoantibodies that activate autophagy via a Fas-dependent pathway in cultured human neuroblastoma SH-Sy5Y cells. METHODS Twenty-five patients with established neurogenic CIP (20 women, 5 men; age range, 21-57 y) were investigated and circulating antineuronal antibodies to enteric neurons were found in 6 (24%) patients. The ability of antineuronal antibodies to induce autophagy was assessed using immunohistochemical, Western immunoblot, and molecular techniques. The presence of autophagosomes was monitored using a specific immunohistochemical marker, anti-microtubule-associated light chain immunoreactivity, and colocalization with mitochondrial- and Fas-activated death domain immunofluorescence using appropriate antibodies in cells exposed to sera from matched healthy controls and patients with neurogenic CIP. RESULTS Exposure of SH-Sy5Y cells to sera from patients with CIP containing antineuronal antibodies revealed increased binding of autoimmune immunoglobulin (IgG class) to the surface of SH-Sy5Y cells and increased formation of autophagosomes showing colocalization with mitochondria and Fas-activated death domain compared with control sera. Pretreatment of sera with either protein L agarose beads or a soluble Fas receptor (extracellular domain) chimera prevented the stimulation of autophagy. CONCLUSIONS We provide novel evidence that antineuronal antibodies may contribute to neuronal dysfunction observed in a subset of patients with neurogenic CIP via autoantibody-mediated activation of autophagy involving the Fas receptor complex.