Estrogen augmented visceral pain and colonic neuron modulation in a double-hit model of prenatal and adult stress.

BACKGROUND: Chronic stress during pregnancy may increase visceral hyperalgesia of offspring in a sex-dependent way. Combining adult stress in offspring will increase this sensitivity. Based on the evidence implicating estrogen in exacerbating visceral hypersensitivity in female rodents in preclinical models, we predicted that chronic prenatal stress (CPS) + chronic adult stress (CAS) will maximize visceral hyperalgesia; and that estrogen plays an important role in colonic hyperalgesia. AIM: The aim was to illuminate the role of estrogen in colonic hyperalgesia and its underlying mechanisms. METHODS: We established a CPS plus CAS rodent model in which the balloon was used to distend the colorectum. The single-fiber recording in vivo and patch clamp experiments in vitro were used to monitor the colonic neuron's activity. The reverse transcription-polymerase chain reaction, western blot, and immunofluorescence were used to study the effects of CPS and CAS on colon primary afferent sensitivity. We used ovariectomy and letrozole to reduce estrogen levels of female rats respectively in order to assess the role of estrogen in female-specific enhanced primary afferent sensitization. RESULTS: Spontaneous activity and single fiber activity were significantly greater in females than in males. The enhanced sensitization in female rats mainly came from low-threshold neurons. CPS significantly increased single-unit afferent fiber activity in L6-S2 dorsal roots in response. Activity was further enhanced by CAS. In addition, the excitability of colon-projecting dorsal root ganglion (DRG) neurons increased in CPS + CAS rats and was associated with a decrease in transient A-type K+ currents. Compared with ovariectomy, treatment with the aromatase inhibitor letrozole significantly reduced estrogen levels in female rats, confirming the gender difference. Moreover, mice treated with letrozole had decreased colonic DRG neuron excitability. The intrathecal infusion of estrogen increased brain-derived neurotrophic factor (BDNF) protein levels and contributed to the response to visceral pain. Western blotting showed that nerve growth factor protein was upregulated in CPS + CAS mice. CONCLUSION: This study adds to the evidence that estrogen-dependent sensitization of primary afferent colon neurons is involved in the development of chronic stress-induced visceral hypersensitivity in female rats.

Estrogen and serotonin enhance stress-induced visceral hypersensitivity in female rats by up-regulating brain-derived neurotrophic factor in spinal cord.

BACKGROUND: We previously reported that female offspring of dams subjected to chronic prenatal stress (CPS) develop enhanced visceral hypersensitivity (VHS) following exposure to chronic stress in adult life that is mediated by up-regulation of spinal cord BDNF. The aims of this study were to examine the roles of estrogen receptor alpha (ERα) and an increase in spinal serotonin signaling in promoting this enhanced VHS in female rats and up-regulation of spinal cord BDNF transcription. METHODS: Pregnant dams were exposed to chronic stress from E11 until delivery. At 8 weeks, a chronic adult stress (CAS) protocol was applied for nine days. KEY RESULTS: Ovariectomy before CAS or treatment with letrozole before and during CAS significantly prevented the development of enhanced VHS in female CPS+CAS rats. Intrathecal application of ERα siRNA significantly reduced VHS, decreased lumbar-sacral spinal cord expression of both ERα and BDNF, and reversed pro-transcriptional epigenetic modifications at BDNF promoter lX. Cerebrospinal fluid serotonin levels and 5HT3A receptor expression in the LS spinal cord were both significantly increased in female CPS+CAS rats. During CAS, intrathecal infusion of alosetron significantly decreased VHS, reduced BDNF and ERα expression in the LS spinal cord, and attenuated RNA pol II and ERα binding to the BNDF core promoter IX. CONCLUSIONS & INFERENCES: Serotonin-mediated activation of 5HT3A receptors in the spinal cord drives the development of enhanced female-specific VHS in our two hit CPS+CAS through up-regulation of spinal cord ERα.

Neonatal Injury Increases Gut Permeability by Epigenetically Suppressing E-Cadherin in Adulthood.

Altered intestinal epithelial integrity is an important susceptibility trait in inflammatory bowel disease (IBD), and early life stressors are reported to contribute to this disease susceptibility in adulthood. To identify disease mechanisms associated with early-life trauma that exacerbate IBD in adulthood, we used a "double-hit" neonatal inflammation (NI) and adult inflammation (AI) model that exhibits more severe mucosal injury in the colon later in life. In this study, we explore the underlying mechanisms of this aggravated injury. In rats exposed to both NI and AI, we found sustained increases in colonic permeability accompanied by significantly attenuated expression of the epithelial junction protein E-cadherin. Quantitative RT-PCR revealed a decreased Cdh1 (gene of E-cadherin) mRNA expression in NI + AI rats compared with NI or AI rats. Next, we performed microRNA microarrays to identify potential regulators of E-cadherin in NI + AI rats. We confirmed the overexpression of miR-155, a predicted regulator of E-cadherin, and selected it for further analysis based on reported significance in human IBD. Using ingenuity pathway analysis software, the targets and related canonical pathway of miR-155 were analyzed. Mechanistic studies identified histone hyperacetylation at the Mir155 promoter in NI + AI rats, concomitant with elevated RNA polymerase II binding. In vitro, E-cadherin knockdown markedly increased epithelial cell permeability, as did overexpression of miR-155 mimics, which significantly suppressed E-cadherin protein. In vivo, NI + AI colonic permeability was significantly reversed with administration of miR-155 inhibitor rectally. Our collective findings indicate that early-life inflammatory stressors trigger a significant and sustained epithelial injury by suppressing E-cadherin through epigenetic mechanisms.

Neonatal Colonic Inflammation Epigenetically Aggravates Epithelial Inflammatory Responses to Injury in Adult Life.

BACKGROUND & AIMS: Early life adversity is considered a risk factor for the development of gastrointestinal diseases, including inflammatory bowel disease. We hypothesized that early life colonic inflammation causes susceptibility to aggravated overexpression of interleukin (IL)1ß. METHODS: We developed a 2-hit rat model in which neonatal inflammation (NI) and adult inflammation (AI) were induced by trinitrobenzene sulfonic acid. RESULTS: Aggravated immune responses were observed in NI + AI rats, including a sustained up-regulation of IL1ß and other cytokines. In parallel with exacerbated loss of inhibitor of kappa B alpha expression, NI + AI rats showed hyperacetylation of histone H4K12 and increased V-Rel Avian Reticuloendotheliosis Viral Oncogene Homolog A binding on the IL1B promoter, accompanied by high levels of norepinephrine/epinephrine. Propranolol, a ß-blocker, markedly ameliorated the inflammatory response and IL1ß overexpression by mitigating against epigenetic modifications. Adrenalectomy abrogated NI-induced disease susceptibility whereas yohimbine sensitized the epithelium for exacerbated immune response. The macrophages of NI rats produced more IL1ß than controls after exposure to lipopolysaccharide (LPS), suggesting hypersensitization; incubation with LPS plus Foradil (Sigma, St. Louis, MO), a ß2-agonist, induced a greater IL1ß expression than LPS alone. Epinephrine and Foradil also exacerbated LPS-induced IL1ß activation in human THP-1-derived macrophages, by increasing acetylated H4K12, and these increases were abrogated by propranolol. CONCLUSIONS: NI sensitizes the colon epithelium for exacerbated IL1ß activation by increasing stress hormones that induce histone hyperacetylation, allowing greater access of nuclear factor-κB to the IL1B promoter and rendering the host susceptible to aggravated immune responses. Our findings suggest that ß blockers have a therapeutic potential for inflammatory bowel disease susceptibility and establish a novel paradigm whereby NI induces epigenetic susceptibility to inflammatory bowel disease.

Noninflammatory upregulation of nerve growth factor underlies gastric hypersensitivity induced by neonatal colon inflammation.

Gastric hypersensitivity is one of the key contributors to the postprandial symptoms of epigastric pain/discomfort, satiety, and fullness in functional dyspepsia patients. Epidemiological studies found that adverse early-life experiences are risk factors for the development of gastric hypersensitivity. Preclinical studies found that neonatal colon inflammation elevates plasma norepinephrine (NE), which upregulates expression of nerve growth factor (NGF) in the muscularis externa of the gastric fundus. Our goal was to investigate the cellular mechanisms by which NE upregulates the expression of NGF in gastric hypersensitive (GHS) rats, which were subjected previously to neonatal colon inflammation. Neonatal colon inflammation upregulated NGF protein, but not mRNA, in the gastric fundus of GHS rats. Western blotting showed upregulation of p110γ of phosphatidylinositol 4,5-bisphosphate 3-kinase (PI3K), phosphoinositide-dependent kinase-1 (PDK1), pAKT(Ser473), and phosphorylated 4E-binding protein (p4E-BP1)(Thr70), suggesting AKT activation and enhanced NGF protein translation. AKT inhibitor MK-2206 blocked the upregulation of NGF in the fundus of GHS rats. Matrix metalloproteinase 9 (MMP-9), the major NGF-degrading protease, was suppressed, indicating that NGF degradation was impeded. Incubation of fundus muscularis externa with NE upregulated NGF by modulating the protein translation and degradation pathways. Yohimbine, an α2-adrenergic receptor antagonist, upregulated plasma NE and NGF expression by activating the protein translation and degradation pathways in naive rats. In contrast, a cocktail of adrenergic receptor antagonists suppressed the upregulation of NGF by blocking the activation of the protein translation and degradation pathways. Our findings provide evidence that the elevation of plasma NE induces NGF expression in the gastric fundus.

Developmental origins of colon smooth muscle dysfunction in IBS-like rats.

Epidemiological studies show that subsets of adult and pediatric patients with irritable bowel syndrome (IBS) have prior exposures to psychological or inflammatory stress. We investigated the cellular mechanisms of colonic smooth muscle dysfunction in adult rats subjected to neonatal inflammation. Ten-day-old male rat pups received 2,4,6-trinitrobenzene sulfonic acid to induce colonic inflammation. Colonic circular smooth muscle strips were obtained 6 to 8 wk later. We found that about half of the neonate pups subjected to inflammatory insult showed a significant increase in expression of the pore-forming α1C-subunit of Cav1.2b channels in adult life. These were the same rats in whom Vip mRNA increased in the colon muscularis externae. Additional experiments showed reduced interaction of histone deacetylase (HDAC) 3 with α1C1b promoter that increased the acetylation of histone H3 lysine 9 (H3K9) in the core promoter region. Vasoactive intestinal peptide (VIP) treatment of naïve muscularis externae swiftly recruited CREB-binding protein (CBP) to the α1C1b promoter and dissociated HDAC3 from this region to initiate transcription. The CBP interaction with the α1C1b promoter was transient, but the dissociation of HDAC3 persisted to sustain H3K9 hyperacetylation and increase in transcription. Intraperitoneal treatment of adult naïve rats with butyrate mimicked the effects of neonatal colon inflammation. We concluded that neonatal inflammation upregulates VIP in the colon muscularis externae, which modulates epigenetic events at the α1C1b promoter to activate α1C1b gene transcription. Inflammatory insult in early life may be one of the etiologies of smooth muscle dysfunction in adult life, which contributes to the altered motility function in patients with diarrhea-predominant IBS.

Paradoxical regulation of ChAT and nNOS expression in animal models of Crohn's colitis and ulcerative colitis.

Morphological and functional changes in the enteric nervous system (ENS) have been reported in inflammatory bowel disease. We examined the effects of inflammation on the expression of choline acetyltransferase (ChAT) and nNOS in the muscularis externae of two models of colonic inflammation, trinitrobenzene sulfonic acid (TNBS)-induced colitis, which models Crohn's disease-like inflammation, and DSS-induced colitis, which models ulcerative Colitis-like inflammation. In TNBS colitis, we observed significant decline in ChAT, nNOS, and protein gene product (PGP) 9.5 protein and mRNA levels. In DSS colitis, ChAT and PGP9.5 were significantly upregulated while nNOS levels did not change. The nNOS dimer-to-monomer ratio decreased significantly in DSS- but not in TNBS-induced colitis. No differences were observed in the percentage of either ChAT (31 vs. 33%)- or nNOS (37 vs. 41%)-immunopositive neurons per ganglia or the mean number of neurons per ganglia (55 ± 5 vs. 59 ± 5, P > 0.05). Incubation of the distal colon muscularis externae in vitro with different types of inflammatory mediators showed that cytokines decreased ChAT and nNOS expression, whereas H2O2, a component of oxidative stress, increased their expression. NF-κB inhibitor MG-132 did not prevent the IL-1ß-induced decline in either ChAT or nNOS expression. These findings showed that TNBS- and DSS-induced inflammation differentially regulates the expression of two critical proteins expressed in the colonic myenteric neurons. These differences are likely due to the exposure of the myenteric plexus neurons to different combinations of Th1-type inflammatory mediators and H2O2 in each model.

Differential immune and genetic responses in rat models of Crohn's colitis and ulcerative colitis.

Crohn's disease and ulcerative colitis are clinically, immunologically, and morphologically distinct forms of inflammatory bowel disease (IBD). However, smooth muscle function is impaired similarly in both diseases, resulting in diarrhea. We tested the hypothesis that differential cellular, genetic, and immunological mechanisms mediate smooth muscle dysfunction in two animal models believed to represent the two diseases. We used the rat models of trinitrobenzene sulfonic acid (TNBS)- and dextran sodium sulfate (DSS)-induced colonic inflammations, which closely mimic the clinical and morphological features of Crohn's disease and ulcerative colitis, respectively. DSS inflammation induced oxidative stress initially in mucosa/submucosa, which then propagated to the muscularis externa to impair smooth muscle function. The muscularis externa showed no increase of cytokines/chemokines. On the other hand, TNBS inflammation almost simultaneously induced oxidative stress, recruited or activated immune cells, and generated cytokines/chemokines in both mucosa/submucosa and muscularis externa. The generation of cytokines/chemokines did not correlate with the recruitment and activation of immune cells. Consequently, the impairment of smooth muscle function in DSS inflammation was primarily due to oxidative stress, whereas that in TNBS inflammation was due to both oxidative stress and proinflammatory cytokines. The impairment of smooth muscle function in DSS inflammation was due to suppression of Gα(q) protein of the excitation-contraction coupling. In TNBS inflammation, it was due to suppression of the α(1C)1b subunit of Ca(v)1.2b channels, CPI-17 and Gα(q). TNBS inflammation increased IGF-1 and TGF-ß time dependently in the muscularis externa. IGF-1 induced smooth muscle hyperplasia; both IGF-1 and TGF-ß induced hypertrophy. In conclusion, both TNBS and DSS induce transmural inflammation, albeit with different types of inflammatory mediators. The recruitment or activation of immune cells does not correlate directly with the intensity of generation of inflammatory mediators. The inflammatory mediators in TNBS and DSS inflammations target different genes to impair smooth muscle function.

Adrenergic stimulation mediates visceral hypersensitivity to colorectal distension following heterotypic chronic stress.

BACKGROUND & AIMS: Chronic stress exacerbates or causes relapse of symptoms such as abdominal pain and cramping in patients with irritable bowel syndrome. We investigated whether chronic stress increases plasma norepinephrine and sensitizes colon-specific dorsal root ganglion (DRG) neurons by increasing expression of nerve growth factor (NGF) in the colon wall. METHODS: Heterotypic chronic stress (HeCS) was applied to male Wistar rats and neurologic and molecular responses were analyzed. Tissues were analyzed for NGF expression. RESULTS: HeCS significantly increased visceromoter response to colorectal distension; expression of NGF increased in colonic muscularis externa and mucosa/submucosa. Rheobase decreased, resting membrane potential was depolarized, and electrogenesis of action potentials increased in colon-specific thoracolumbar DRG neurons. Luminal administration of resiniferatoxin in distal colon, systemic administration of anti-NGF antibody, or inhibition of the NGF receptor trkA by k252a or antisense oligonucleotides in thoracolumbar DRG blocked the chronic stress-induced visceral hypersensitivity to colorectal distension. Blockade of alpha1/alpha2- and beta1/beta2-adrenergic receptors prevented the stress-induced visceral hypersensitivity and increased expression of NGF in the colon wall. HeCS did not induce any inflammatory response in the colon wall. CONCLUSIONS: The peripheral stress mediator norepinephrine induces visceral hypersensitivity to colorectal distension in response to HeCS by increasing the expression of NGF in the colon wall, which sensitizes primary afferents in the absence of an inflammatory response.

The endogenous hydrogen sulfide producing enzyme cystathionine-beta synthase contributes to visceral hypersensitivity in a rat model of irritable bowel syndrome.

BACKGROUND: The pathogenesis of visceral hypersensitivity, a characteristic pathophysiological feature of irritable bowel syndrome (IBS), remains elusive. Recent studies suggest a role for hydrogen sulfide (H2S) in pain signaling but this has not been well studied in visceral models of hyperalgesia. We therefore determined the role for the endogenous H2S producing enzyme cystathionine-beta-synthetase (CBS) in a validated rat model of IBS-like chronic visceral hyperalgesia (CVH). CVH was induced by colonic injection of 0.5% acetic acid (AA) in 10-day-old rats and experiments were performed at 8-10 weeks of age. Dorsal root ganglion (DRG) neurons innervating the colon were labeled by injection of DiI (1,1'-dioleyl-3,3,3',3-tetramethylindocarbocyanine methanesulfonate) into the colon wall. RESULTS: In rat DRG, CBS-immunoreactivity was observed in approximately 85% of predominantly small- and medium-sized neurons. Colon specific DRG neurons revealed by retrograde labeling DiI were all CBS-positive. CBS-positive colon neurons co-expressed TRPV1 or P2X3 receptors. Western blotting analysis showed that CBS expression was significantly increased in colon DRGs 8 weeks after neonatal AA-treatment. Furthermore, the CBS inhibitor hydroxylamine markedly attenuated the abdominal withdrawal reflex scores in response to colorectal distention in rats with CVH. By contrast, the H2S donor NaHS significantly enhanced the frequency of action potentials of colon specific DRG neurons evoked by 2 times rheobase electrical stimulation. CONCLUSION: Our results suggest that upregulation of CBS expression in colonic DRG neurons and H2S signaling may play an important role in developing CVH, thus identifying a specific neurobiological target for the treatment of CVH in functional bowel syndromes.

A computerized analysis of robotic versus laparoscopic task performance.

INTRODUCTION: Robotic technology has been postulated to improve performance in advanced surgical skills. We utilized a novel computerized assessment system to objectively describe the technical enhancement in task performance comparing robotic and laparoscopic instrumentation. METHODS AND PROCEDURES: Advanced laparoscopic surgeons (2-10 yrs experience) performed three unique task modules using laparoscopic and Telerobotic surgical instrumentation (Intuitive Surgical, Sunnyvale, CA). Performance was evaluated using a computerized assessment system (ProMIS, Dublin, Ireland) and results were recorded as time (s), path (mm) and precision. Each surgeon had an initial training session followed by two testing sessions for each module. A paired Student's t-test was used to analyze the data. RESULTS: Ten surgeons completed the study. 8/10 surgeons had significant technical enhancement utilizing robotic technology. CONCLUSIONS: The ProMIS computerized assessment system can be modified to objectively obtain task performance data with robotic instrumentation. All the tasks were performed faster and with more precision using the robotic technology than standard laparoscopy.

Hirschsprung's disease in a young adult: report of a case and review of the literature.

Hirschsprung's disease (HD) in adults is rare and often undiagnosed or misdiagnosed. We report a case of HD in a 26-year-old woman who had a history of chronic constipation that required laxatives and enemas since early childhood. She developed severe intestinal obstruction and presented to the emergency department with significant abdominal distension. A computed tomographic scan confirmed significant fecal loading of the entire colon and rectum. An anal manometry revealed lack of normal rectoanal inhibitory reflex. A rectal biopsy showed hypoganglionic anorectum, suspicious for HD. Because of the severe fecal retention that was refractory to conservative management, total proctocolectomy with ileal pouch-anal anastomosis was performed. The entire colon showed massive dilatation and marked wall thickening. Histologic examination showed absence of ganglion cells in submucosal (Meissner's) and myenteric (Auerbach's) plexuses in the distal rectum. A diagnosis of adult HD was made. Her postoperative course was uneventful with complete resolution of the symptoms. Hirschsprung's disease should be considered in adults who have long-standing and refractory constipation.

Trypsin mediates nociception via the proteinase-activated receptor 2: a potentially novel role in pancreatic pain.

BACKGROUND & AIMS: The pathogenesis of pain in pancreatitis remains poorly understood. We hypothesized that trypsin, a key inflammatory mediator in this condition, can also activate nociceptive neurons via the proteinase-activated receptor 2. METHODS: Double immunohistochemical staining of T8 to T12 dorsal root ganglia sections was performed with antibodies against proteinase-activated receptor 2 and vanilloid receptor 1, a marker for primary nociceptive neurons. In vivo nociceptive activity was measured by FOS immunoreactivity in thoracic spinal dorsal horn segments after intrapancreatic administration of proteinase-activated receptor 2 agonists. Pain behavior was assessed by visceromotor reflex activity in response to noxious stimulation of the pancreas with proteinase-activated receptor 2 agonists. RESULTS: Proteinase-activated receptor 2 was expressed by virtually all nociceptive neurons in thoracic dorsal root ganglia. Intraductal trypsin, in subinflammatory concentrations, activated spinal dorsal horn neurons in a dose-dependent manner, as measured by FOS expression. Both trypsin and a proteinase-activated receptor 2-specific peptide agonist induced a behavioral pain response when infused into the pancreatic duct of awake rats. Preinfusion of the pancreatic duct with proteinase-activated receptor 2-specific activating peptide desensitized the response to trypsin. CONCLUSIONS: Our findings suggest a novel proteinase-activated receptor 2-mediated role for trypsin in the pathogenesis of pancreatic pain and one that is independent of its inflammatory effect.

Acute pancreatitis results in referred mechanical hypersensitivity and neuropeptide up-regulation that can be suppressed by the protein kinase inhibitor k252a.

Although pain is a cardinal feature of pancreatitis, its pathogenesis is poorly understood and treatment remains difficult. Nociceptive sensitization in several somatic pain models has been associated with activation of protein kinases including trkA, protein kinase C, and protein kinase A. We therefore tested the hypothesis that systemic treatment with a kinase inhibitor, k252a, known to inhibit all of these kinases would alleviate pain in an animal model of pancreatitis. Von Frey filament testing of somatic referral regions was evaluated as a method to measure referred pain in a rat model of acute necrotizing pancreatitis induced by L-arginine. Rats with pancreatitis showed increased sensitivity to abdominal stimulation with Von Frey filament. This referred mechanical sensitivity was associated with an 8-fold increase in levels of phosphorylated trkA in the pancreas and with significant up-regulation of both calcitonin gene-related peptide and preprotachykinin mRNA expression in thoracic dorsal root ganglia and with increased calcitonin gene-related peptide and substance P immunoreactivity in spinal cord segment T10. Treatment with the kinase inhibitor k252a suppressed the phosphorylation of trkA in the pancreas as well as reversed both the behavioral changes and the increase in neuropeptide expression associated with pancreatitis.