Liansheng Liu

Nerve growth factor modulates TRPV1 expression and function and mediates pain in chronic pancreatitis.

BACKGROUND & AIMS The pathogenesis of pain in chronic pancreatitis (CP) is poorly understood and treatment remains difficult. We hypothesized that nerve growth factor (NGF) plays a key role in this process via its effects on the transient receptor potential vanilloid 1, TRPV1. METHODS CP was induced by intraductal injection of trinitrobenzene sulfonic acid in rats. After 3 weeks, anti-NGF antibody or control serum was administered daily for 1 week. Pancreatic hyperalgesia was assessed by nocifensive behavioral response to electrical stimulation of the pancreas as well as by referred somatic pain assessed by von Frey filament testing. TRPV1 currents in pancreatic sensory neurons were examined by patch-clamp. The expression and function of TRPV1 in pancreas-specific nociceptors was examined by immunostaining and quantification of messenger RNA levels. RESULTS Blockade of NGF significantly attenuated pancreatic hyperalgesia and referred somatic pain compared with controls. It also decreased TRPV1 current density and open probability and reduced the proportion of pancreatic sensory neurons that expressed TRPV1 as well as levels of TRPV1 in these neurons. CONCLUSIONS These findings emphasize a key role for NGF in pancreatic pain and highlight the role it plays in the modulation of TRPV1 expression and activity in CP.

Transient Gastric Irritation in the Neonatal Rats Leads to Changes in Hypothalamic CRF Expression, Depression- and Anxiety-Like Behavior as Adults

Aims A disturbance of the brain-gut axis is a prominent feature in functional bowel disorders (such as irritable bowel syndrome and functional dyspepsia) and psychological abnormalities are often implicated in their pathogenesis. We hypothesized that psychological morbidity in these conditions may result from gastrointestinal problems, rather than causing them. Methods Functional dyspepsia was induced by neonatal gastric irritation in male rats. 10-day old male Sprague-Dawley rats received 0.1% iodoacetamide (IA) or vehicle by oral gavage for 6 days. At 8–10 weeks of age, rats were tested with sucrose preference and forced-swimming tests to examine depression-like behavior. Elevated plus maze, open field and light-dark box tests were used to test anxiety-like behaviors. ACTH and corticosterone responses to a minor stressor, saline injection, and hypothalamic CRF expression were also measured. Results Behavioral tests revealed changes of anxiety- and depression-like behaviors in IA-treated, but not control rats. As compared with controls, hypothalamic and amygdaloid CRF immunoreactivity, basal levels of plasma corticosterone and stress-induced ACTH were significantly higher in IA-treated rats. Gastric sensory ablation with resiniferatoxin had no effect on behaviors but treatment with CRF type 1 receptor antagonist, antalarmin, reversed the depression-like behavior in IA-treated rats Conclusions The present results suggest that transient gastric irritation in the neonatal period can induce a long lasting increase in depression- and anxiety-like behaviors, increased expression of CRF in the hypothalamus, and an increased sensitivity of HPA axis to stress. The depression-like behavior may be mediated by the CRF1 receptor. These findings have significant implications for the pathogenesis of psychological co-morbidity in patients with functional bowel disorders.

Substance P and Calcitonin Gene Related Peptide Mediate Pain in Chronic Pancreatitis and Their Expression is Driven by Nerve Growth Factor

CONTEXT Calcitonin gene-related peptide (CGRP), substance P and nerve growth factor play an important role in inflammatory pain in various somatic pain models but their role in chronic pancreatitis has not been well studied. OBJECTIVES The aim of this study was to investigate the effects of intrathecal administration of calcitonin gene-related peptide antagonist and substance P receptor antagonist on pain behavior in a rat model of chronic pancreatitis and to determine whether nerve growth factor drives the up-regulation of expression of these neuropeptides in sensory neurons. METHODS Pancreatitis was induced by retrograde infusion of trinitobenzene sulfonic acid into the pancreatic duct of adult rats. Three weeks post infusion continuous intrathecal infusion of the calcitonin gene-related peptide antagonist alpha CGRP8-37 or neurokinin-1 receptor antagonist CP-96345 or its inactive enantiomer CP-96344 was administered for seven days. The effects of treatment on pancreatic hyperalgesia were assessed by sensitivity of the abdominal wall to von Frey filament probing as well as by the nocifensive response to electrical stimulation of the pancreas. In a separate experiment chronic pancreatitis was induced and pancreas specific dorsal root ganglion neurons labeled with DiI were assessed for calcitonin gene-related peptide and substance P immunoreactivity. RESULTS Intrathecal infusion of calcitonin gene-related peptide and neurokinin-1 receptor antagonists significantly attenuated behavioral pain responses in rats with chronic pancreatitis. Further, treatment of chronic pancreatitis rats with nerve growth factor antibody significantly reduced pancreas specific neurons expressing calcitonin gene-related peptide and substance P in thoracic dorsal root ganglion. CONCLUSIONS Calcitonin gene-related peptide and substance P mediate pancreatic hyperalgesia in chronic pancreatitis and nerve growth factor in turn sustains the up-regulation of these neuropeptides in pancreatic sensory neurons.

The analgesic effects of the GABAB receptor agonist, baclofen, in a rodent model of functional dyspepsia.

Background  The amino acid γ‐aminobutyric acid (GABA) is an important modulator of pain but its role in visceral pain syndromes is just beginning to be studied. Our aims were to investigate the effect and mechanism of action of the GABAB receptor agonist, baclofen, on gastric hypersensitivity in a validated rat model of functional dyspepsia (FD).

Adult enteric nervous system in health is maintained by a dynamic balance between neuronal apoptosis and neurogenesis

Significance The demonstration of a robust neurogenesis program in the adult gut and the existence of an enteric neural precursor cell (ENPC) responsible for the same has profound biological and clinical implications. This demonstrates the presence of robust adult neurogenesis outside of the CNS, and indicates the vulnerability of the enteric nervous system to exogenous influences, even in adults. As an example, it is possible that acquired diseases of the enteric nervous system, such as achalasia, may result from a loss of ENPC, analogous to congenital disorders, such as Hirschsprung’s. The ability to identify the adult ENPC will therefore enable a new understanding of the pathogenesis of enteric neuromuscular diseases as well as the development of novel regenerative therapies. According to current dogma, there is little or no ongoing neurogenesis in the fully developed adult enteric nervous system. This lack of neurogenesis leaves unanswered the question of how enteric neuronal populations are maintained in adult guts, given previous reports of ongoing neuronal death. Here, we confirm that despite ongoing neuronal cell loss because of apoptosis in the myenteric ganglia of the adult small intestine, total myenteric neuronal numbers remain constant. This observed neuronal homeostasis is maintained by new neurons formed in vivo from dividing precursor cells that are located within myenteric ganglia and express both Nestin and p75NTR, but not the pan-glial marker Sox10. Mutation of the phosphatase and tensin homolog gene in this pool of adult precursors leads to an increase in enteric neuronal number, resulting in ganglioneuromatosis, modeling the corresponding disorder in humans. Taken together, our results show significant turnover and neurogenesis of adult enteric neurons and provide a paradigm for understanding the enteric nervous system in health and disease.

Systemic administration of anti-NGF increases A-type potassium currents and decreases pancreatic nociceptor excitability in a rat model of chronic pancreatitis.

We have previously shown that pancreatic sensory neurons in rats with chronic pancreatitis (CP) display increased excitability associated with a decrease in transient inactivating potassium currents (I(A)), thus accounting in part for the hyperalgesia associated with this condition. Because of its well known role in somatic hyperalgesia, we hypothesized a role for the nerve growth factor (NGF) in driving these changes. CP was induced by intraductal injection of trinitrobenzene sulfonic acid (TNBS) in rats. After 3 wk, anti-NGF antibody or control serum was injected intra-peritoneally daily for 1 wk. This protocol was repeated in another set of experiments in control rats (receiving intraductal PBS instead of TNBS). Pancreatic nociceptors labeled with the dye Dil were identified, and patch-clamp recordings were made from acutely dissociated DRG neurons. Sensory neurons from anti-NGF-treated rats displayed a lower resting membrane potential, increased rheobase, decreased burst discharges in response to stimulatory current, and decreased input resistance compared with those treated with control serum. Under voltage-clamp condition, neuronal I(A) density was increased in anti-NGF-treated rats compared with rats treated with control serum. However, anti-NGF treatment had no effect on electrophysiological parameters in neurons from control rats. The expression of Kv-associated channel or ancillary genes Kv1.4, 4.1, 4.2, 4.3, and DPP6, DPP10, and KCHIPs 1-4 in pancreas-specific nociceptors was examined by laser-capture microdissection and real-time PCR quantification of mRNA levels. No significant differences were seen among those. These findings emphasize a key role for NGF in maintaining neuronal excitability in CP specifically via downregulation of I(A) by as yet unknown mechanisms.

Anatomical and functional characterization of a duodeno-pancreatic neural reflex that can induce acute pancreatitis

Neural cross talk between visceral organs may play a role in mediating inflammation and pain remote from the site of the insult. We hypothesized such a cross talk exists between the duodenum and pancreas, and further it induces pancreatitis in response to intraduodenal toxins. A dichotomous spinal innervation serving both the duodenum and pancreas was examined, and splanchnic nerve responses to mechanical stimulation of these organs were detected. This pathway was then excited on the duodenal side by exposure to ethanol followed by luminal mustard oil to activate transient receptor potential subfamily A, member 1 (TRPA1). Ninety minutes later, pancreatic inflammation was examined. Ablation of duodenal afferents by resiniferatoxin (RTX) or blocking TRPA1 by Chembridge (CHEM)-5861528 was used to further investigate the duodeno-pancreatic neural reflex via TRPA1. ~40% of dorsal root ganglia (DRG) from the spinal cord originated from both duodenum and pancreas via dichotomous peripheral branches; ~50% splanchnic nerve single units responded to mechanical stimulation of both organs. Ethanol sensitized TRPA1 currents in cultured DRG neurons. Pancreatic edema and myeloperoxidase activity significantly increased after intraduodenal ethanol followed by mustard oil (but not capsaicin) but significantly decreased after ablation of duodenal afferents by using RTX or blocking TRPA1 by CHEM-5861528. We found the existence of a neural cross talk between the duodenum and pancreas that can promote acute pancreatitis in response to intraduodenal chemicals. It also proves a previously unexamined mechanism by which alcohol can induce pancreatitis, which is novel both in terms of the site (duodenum), process (neurogenic), and receptor (TRPA1).

Brain-derived neurotrophic factor is upregulated in rats with chronic pancreatitis and mediates pain behavior.

Objectives: We examined the role of brain-derived neurotrophic factor (BDNF) in the pathogenesis of pain in an experimental model of chronic pancreatitis (CP). Methods: Pancreatitis was induced by retrograde infusion of trinitrobenzene sulfonic acid into the pancreatic duct of adult rats. Twenty-one days after injection, BDNF expression was examined in pancreas-specific dorsal root ganglia (DRGs) by immunohistochemistry, and protein levels were quantified from DRGs and spinal cord extracts. The effects of intrathecal infusion of a neutralizing antibody to BDNF on pancreatic hyperalgesia were assessed by the sensitivity of the abdominal wall to filament probing as well as the nocifensive behavior to electrical stimulation of the pancreas. Results: Levels of BDNF in DRGs and spinal cords (T9-13) were significantly higher in trinitrobenzene sulfonic acid rats compared with controls, accompanied by an increase in the number of pancreas-specific neurons expressing BDNF immunoreactivity. Brain-derived neurotrophic factor antagonism suppressed phospho-tropomyosin-related kinase B receptor levels in the spinal cord and significantly reduced behavioral responses in rats with CP. Conclusions: Brain-derived neurotrophic factor is upregulated in pancreas-specific primary afferent neurons in rats with CP, and BDNF antagonism is associated with a reduction of pain-related behavior in these animals, suggesting an important role for this neurotransmitter in the nociception of CP.

Imaging Macrophage Accumulation in a Murine Model of Chronic Pancreatitis with [125I]iodoDPA SPECT-CT

Pancreatitis remains a diagnostic challenge in patients with mild to moderate disease, with current imaging modalities being inadequate. Given the prominent macrophage infiltration in chronic pancreatitis, we hypothesized that 125I-iodo-DPA-713, a small-molecule radiotracer that specifically targets macrophages, could be used with SPECT/CT to image pancreatic inflammation in a relevant experimental model. Methods: Chronic pancreatitis was induced with cerulein in C57BL/6 mice, which were contrasted with saline-injected control mice. The animals were imaged at 7 wk after induction using N,N-diethyl-2-(2-(3-125I-iodo-4-methoxyphenyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl)acetamide (125I-iodo-DPA-713) SPECT/CT or 18F-FDG PET/CT. The biodistribution of 125I-iodo-DPA-713 was determined under the same conditions, and a pair of mice was imaged using a fluorescent analog of 125I-iodo-DPA-713, DPA-713-IRDye800CW, for correlative histology. Results: Pancreatic 125I-iodo-DPA-713 uptake was significantly higher in treated mice than control mice (5.17% ± 1.18% vs. 2.41% ± 0.34% injected dose/g, P = 0.02), as corroborated by imaging. Mice imaged with 18F-FDG PET/CT showed cerulein-enhanced pancreatic uptake in addition to a moderate signal from healthy pancreas. Near-infrared fluorescence imaging with DPA-713-IRDye800CW showed strong pancreatic uptake, focal liver uptake, and gastrointestinal uptake in the treated mice, whereas the control mice showed only urinary excretion. Ex vivo fluorescence microscopy revealed a large influx of macrophages in the pancreas colocalizing with the retained fluorescent probe in the treated but not the control mice. Conclusion: These data support the application of both 125I-iodo-DPA-713 SPECT/CT and DPA-713-IRDye800CW near-infrared fluorescence to delineate pancreatic, liver, or intestinal inflammation in living mice.

Mo2037 Phosphodiesterase 2A Mediate Neonatal Inflammation-Induced Visceral Hypersensitivity in a Mouse Model of Irritable Bowel Syndrome

G A A b st ra ct s in each disease type. Figure 1 shows the number of differentially regulated transcripts in various comparisons. IBS vs CD showed the highest number of deregulated transcripts (N= 686). Table 1 shows GO terms, associated genes and p values inferred from functional annotation clustering for all the comparisons. Clustering revealed upregulation of inflammatory genes (e.g., CD38molecule; TRGC2: T-cell receptor gamma locus; IGHG4: Immunoglobulin heavy constant gamma 4 (G4M marker) for both CD and UC compared to IBS. There were 4 transcripts deregulated between IBS and HCs. microRNA miR-4461 was significantly downregulated (FC: -2.2, p=0.006) in IBS compared to HCs. However, at a lower fold change cutoff (FC>1.3), inflammation and defense response associated genes, such as NFKB1A, S100 Calcium Binding Protein A12 (S100A12), NCF1B, were upregulated in IBS patients vs. HCs. miR-27A, reported as a class of adipogenic inhibitors, was upregulated in IBS patients. Most genes that had significantly different expressions in IBS vs. HCs were unannotated. Conclusions:Our findings suggest that while inflammatory genes were modestly upregulated in IBS vs. HCs, they were much lower compared to IBD. Interestingly, miR-27, which is upregulated in obesity, is also upregulated in IBS, suggesting that there may be a shared pathophysiologic mechanism. Thus, comparing mRNA profiles of IBS to that of GI disease controls such as IBD and healthy controls may lead to a better understanding of the pathophysiology of IBS and potentially a diagnostic biomarker. Gene Ontology (GO) Terms Associated with IBS vs Healthy and Disease Controls