Li-Ya Qiao

Differential changes in brain-derived neurotrophic factor and extracellular signal-regulated kinase in rat primary afferent pathways with colitis

Abstract  Brain‐derived neurotrophic factor (BDNF) has been postulated to participate in inflammation‐induced visceral hypersensitivity by modulating the sensitivity of visceral afferents through the activation of intracellular signalling pathways such as the extracellular signal‐regulated kinase (ERK) pathway. In the current study, we assessed the expression levels of BDNF and phospho‐ERK in lumbosacral dorsal root ganglia (DRG) and spinal cord before and during tri‐nitrobenzene sulfonic acid (TNBS)‐induced colitis in rats with real‐time PCR, ELISA, western blot and immunohistochemical techniques. BDNF mRNA and protein levels were increased in L1 and S1 but not L6 DRG when compared with control (L1: two‐ to five‐fold increases, P < 0.05; S1: two‐ to three‐fold increases, P < 0.05); however, BDNF protein but not mRNA level was increased in L1 and S1 spinal cord when compared with control. In parallel, TNBS colitis significantly induced phospho‐ERK1/2 expression in L1 (four‐ to five‐fold, P < 0.05) and S1 (two‐ to three‐fold, P < 0.05) but not in L6 spinal cord levels. Immunohistochemistry results showed that the increase in phospho‐ERK1/2 expression occurred at the region of the superficial dorsal horn and grey commisure of the spinal cord. In contrast, there was no change in phospho‐ERK5 in any level of the spinal cord examined during colitis. The regional and time‐specific changes in the levels of BDNF mRNA, protein and phospho‐ERK with colitis may be a result of increased transcription of BDNF in DRG and anterograde transport of BDNF from DRG to spinal cord where it activates intracellular signalling molecules such as ERK1/2.

Endogenous Nerve Growth Factor Regulates Collagen Expression and Bladder Hypertrophy through Akt and MAPK Pathways during Cystitis

Type I collagen forms the main constituent of the extracellular matrix in visceral organs. We reported here that cyclophosphamide (CYP)-induced cystitis significantly increased the production of type I collagen in the inflamed bladder leading to increases in the bladder weight and the thickness of the bladder wall. The endogenous nerve growth factor (NGF) in the urinary bladder regulated type I collagen expression because the neutralizing NGF antibody attenuated cystitis-induced type I collagen up-regulation in the inflamed bladder. Neutralizing NGF antibody also subsequently reversed cystitis-induced increases in bladder weight. Further studies on the intermediate signaling pathways mediating NGF-induced type I collagen expression in the inflamed bladder during cystitis revealed that Akt, JNK, and ERK1/2 activities were increased in the inflamed bladder, whereas p38 MAPK remained unchanged. Suppression of endogenous NGF level with neutralizing NGF antibody significantly blocked the increased activity of Akt, JNK, and ERK1/2 in the inflamed bladder during cystitis. These results indicate that endogenous NGF plays an important role in the activation of Akt and MAPK in the urinary bladder and in bladder hypertrophy during cystitis.

Up-regulation of brain-derived neurotrophic factor in primary afferent pathway regulates colon-to-bladder cross-sensitization in rat

BackgroundIn humans, inflammation of either the urinary bladder or the distal colon often results in sensory cross-sensitization between these organs. Limited information is known about the mechanisms underlying this clinical syndrome. Studies with animal models have demonstrated that activation of primary afferent pathways may have a role in mediating viscero-visceral cross-organ sensitization.MethodsColonic inflammation was induced by a single dose of tri-nitrobenzene sulfonic acid (TNBS) instilled intracolonically. The histology of the colon and the urinary bladder was examined by hematoxylin and eosin (H&E) stain. The protein expression of transient receptor potential (TRP) ion channel of the vanilloid type 1 (TRPV1) and brain-derived neurotrophic factor (BDNF) were examined by immunohistochemistry and/or western blot. The inter-micturition intervals and the quantity of urine voided were obtained from analysis of cystometrograms.ResultsAt 3 days post TNBS treatment, the protein level of TRPV1 was increased by 2-fold (p < 0.05) in the inflamed distal colon when examined with western blot. TRPV1 was mainly expressed in the axonal terminals in submucosal area of the distal colon, and was co-localized with the neural marker PGP9.5. In sensory neurons in the dorsal root ganglia (DRG), BDNF expression was augmented by colonic inflammation examined in the L1 DRG, and was expressed in TRPV1 positive neurons. The elevated level of BDNF in L1 DRG by colonic inflammation was blunted by prolonged pre-treatment of the animals with the neurotoxin resiniferatoxin (RTX). Colonic inflammation did not alter either the morphology of the urinary bladder or the expression level of TRPV1 in this viscus. However, colonic inflammation decreased the inter-micturition intervals and decreased the quantities of urine voided. The increased bladder activity by colonic inflammation was attenuated by prolonged intraluminal treatment with RTX or treatment with intrathecal BDNF neutralizing antibody.ConclusionAcute colonic inflammation increases bladder activity without affecting bladder morphology. Primary afferent-mediated BDNF up-regulation in the sensory neurons regulates, at least in part, the bladder activity during colonic inflammation.

Endogenous IGF-I and αvβ3 integrin ligands regulate increased smooth muscle growth in TNBS-induced colitis

Endogenous insulin-like growth factor-I (IGF-I) regulates intestinal smooth muscle growth by concomitantly stimulating proliferation and inhibiting apoptosis. IGF-I-stimulated growth is augmented by the alpha(v)beta(3) integrin ligands vitronectin and fibronectin. IGF-I expression in smooth muscle is increased in both TNBS-induced colitis and Crohns disease. We hypothesized that intestinal inflammation increased vitronectin and fibronectin expression by smooth muscle and, along with IGF-I upregulation, increased intestinal muscle growth. Intestinal smooth muscle cells were examined 7 days following the induction of TNBS-induced colitis. Although alpha(v)beta(3) integrin expression was not altered by TNBS-induced colitis, vitronectin and fibronectin levels were increased by 80 +/- 10% and 90 +/- 15%, above control levels, respectively. Basal IGF-I receptor phosphorylation in inflamed muscle from TNBS-treated rats was increased by 86 +/- 8% over vehicle-treated controls. Basal ERK1/2, p70S6 kinase, and GSK-3beta phosphorylation in muscle cells of TNBS-treated rats were also increased by 140-180%. TNBS treatment increased basal muscle cell proliferation by 130 +/- 15% and decreased apoptosis by 20 +/- 2% compared with that in vehicle-treated controls. The changes in proliferation and apoptosis were reversed by an IGF-I receptor tyrosine kinase inhibitor or an alpha(v)beta(3) integrin antagonist. The results suggest that smooth muscle hyperplasia in TNBS-induced colitis partly results from the upregulation of endogenous IGF-I and ligands of alpha(v)beta(3) integrin that mediate increased smooth muscle cell proliferation and decreased apoptosis. This paper has identified one mechanism regulating smooth muscle hyperplasia, a feature of stricture formation that occurs in the chronically inflamed intestine of TNBS-induced colitis and potentially Crohns disease.

Colitis elicits differential changes in the expression levels of receptor tyrosine kinase TrkA and TrkB in colonic afferent neurons: A possible involvement of axonal transport

&NA; The role of nerve growth factor (NGF) and brain‐derived neurotrophic factor (BDNF) in colitis‐induced hypersensitivity has been suggested. NGF and BDNF facilitate cellular physiology through binding to receptor tyrosine kinase TrkA and TrkB, respectively. The present study by examining the mRNA and/or protein levels of TrkA and TrkB in the distal colon and in colonic primary afferent neurons in the dorsal root ganglia (DRG) during colitis demonstrated that colitis elicited location‐specific changes in the mRNA and protein levels of TrkA and TrkB in colonic primary sensory pathways. In colitis both the TrkA and TrkB protein levels were increased in the L1 and S1 DRGs in a time‐dependent manner; however, the level of TrkB mRNA but not TrkA mRNA was increased in these DRGs. Further experiments showed that colitis facilitated a retrograde transport of TrkA protein toward and an anterograde transport of TrkA mRNA away from the DRG, which may contribute to the increased TrkA mRNA level in the distal colon during colitis. Colitis also increased the level of NGF mRNA but not BDNF mRNA in the distal colon. Double staining showed that the expression of TrkA but not TrkB was increased in the specifically labeled colonic afferent neurons in the L1 and S1 DRGs during colitis; this increase in TrkA level was attenuated by pretreatment with resiniferatoxin. These results suggested that colitis‐induced primary afferent activation involved retrograde transport of TrkA but not TrkB from the distal colon to primary afferent neurons in DRG.

Endogenous PI3K/Akt and NMDAR act independently in the regulation of CREB activity in lumbosacral spinal cord in cystitis ☆

The integral interaction of signaling components in the regulation of visceral inflammation-induced central sensitization in the spinal cord has not been well studied. Here we report that phosphoinositide 3-kinase (PI3K)-dependent Akt activation and N-methyl-d-aspartic acid receptor (NMDAR) in lumbosacral spinal cord independently regulate the activation of cAMP response element-binding protein (CREB) in vivo in a rat visceral pain model of cystitis induced by intraperitoneal injection of cyclophosphamide (CYP). We demonstrate that suppression of endogenous PI3K/Akt activity with a potent PI3K inhibitor LY294002 reverses CYP-induced phosphorylation of CREB, however, it has no effect on CYP-induced phosphorylation of NR1 at Ser(897) and Ser(896); conversely, inhibition of NMDAR in vivo with MK801 fails to block CYP-induced Akt activation but significantly attenuates CYP-induced CREB phosphorylation in lumbosacral spinal cord. This novel interrelationship of PI3K/Akt, NMDAR, and CREB activation in lumbosacral spinal cord is further confirmed in an ex vivo spinal slice culture system exposed to an excitatory neurotransmitter calcitonin gene-related peptide (CGRP). Consistently we found that CGRP-triggered CREB activation can be blocked by both PI3K inhibitor LY294002 and NMDAR antagonists MK801 and D-AP5. However, CGRP-triggered Akt activation cannot be blocked by MK801 or D-AP5; vice versa, LY294002 pretreatment that suppresses the Akt activity fails to reverse CGRP-elicited NR1 phosphorylation. These results suggest that PI3K/Akt and NMDAR independently regulate spinal plasticity in visceral pain model, and target of a single pathway is necessary but not sufficient in treatment of visceral hypersensitivity.

Up-regulation of brain-derived neurotrophic factor is regulated by extracellular signal-regulated protein kinase 5 and by nerve growth factor retrograde signaling in colonic afferent neurons in colitis

Brain-derived neurotrophic factor (BDNF) plays an essential role in sensory neuronal activation in response to visceral inflammation. Here we report that BDNF up-regulation in the primary afferent neurons in the dorsal root ganglia (DRG) in a rat model of colitis is mediated by the activation of endogenous extracellular signal-regulated protein kinase (ERK) 5 and by nerve growth factor (NGF) retrograde signaling. At 7 days of colitis, the expression level of BDNF is increased in conventional neuronal tracing dye Fast Blue labeled primary afferent neurons that project to the distal colon. In these neurons, the phosphorylation (activation) level of ERK5 is also increased. In contrast, the level of phospho-ERK1/2 is not changed in the DRG during colitis. Prevention of the ERK5 activation in vivo with an intrathecal application of the MEK inhibitor PD98059 significantly attenuates the colitis-induced increases in BDNF expression in the DRG. Further studies show that BDNF up-regulation in the DRG is triggered by NGF retrograde signaling which also involves activation of the MEK/ERK pathways. Application of exogenous NGF exclusively to the compartment containing DRG nerve terminals in an ex vivo ganglia-nerve preparation markedly increases the BDNF expression level in the DRG neuronal cell body that is placed in a different compartment; this BDNF elevation is attenuated by U0126, PD98059 and a specific ERK5 inhibitor BIX02188. These results demonstrate the mechanisms and pathways by which BDNF expression is elevated in primary sensory neurons following visceral inflammation that is mediated by increased activity of ERK5 and is likely to be triggered by the elevated NGF level in the inflamed viscera.

Activation of extracellular signal-regulated protein kinase 5 is essential for cystitis- and nerve growth factor-induced calcitonin gene-related peptide expression in sensory neurons

BackgroundCystitis causes considerable neuronal plasticity in the primary afferent pathways. The molecular mechanism and signal transduction underlying cross talk between the inflamed urinary bladder and sensory sensitization has not been investigated.ResultsIn a rat cystitis model induced by cyclophosphamide (CYP) for 48 h, the mRNA and protein levels of the excitatory neurotransmitter calcitonin gene-related peptide (CGRP) are increased in the L6 dorsal root ganglia (DRG) in response to bladder inflammation. Cystitis-induced CGRP expression in L6 DRG is triggered by endogenous nerve growth factor (NGF) because neutralization of NGF with a specific NGF antibody reverses CGRP up-regulation during cystitis. CGRP expression in the L6 DRG neurons is also enhanced by retrograde NGF signaling when NGF is applied to the nerve terminals of the ganglion-nerve two-compartmented preparation. Characterization of the signaling pathways in cystitis- or NGF-induced CGRP expression reveals that the activation (phosphorylation) of extracellular signal-regulated protein kinase (ERK)5 but not Akt is involved. In L6 DRG during cystitis, CGRP is co-localized with phospho-ERK5 but not phospho-Akt. NGF-evoked CGRP up-regulation is also blocked by inhibition of the MEK/ERK pathway with specific MEK inhibitors U0126 and PD98059, but not by inhibition of the PI3K/Akt pathway with inhibitor LY294002. Further examination shows that cystitis-induced cAMP-responsive element binding protein (CREB) activity is expressed in CGRP bladder afferent neurons and is co-localized with phospho-ERK5 but not phospho-Akt. Blockade of NGF action in vivo reduces the number of DRG neurons co-expressing CGRP and phospho-CREB, and reverses cystitis-induced increases in micturition frequency.ConclusionsA specific pathway involving NGF-ERK5-CREB axis plays an essential role in cystitis-induced sensory activation.

Prolonged sympathetic innervation of sensory neurons in rat thoracolumbar dorsal root ganglia during chronic colitis

Background  Peripheral irritation‐induced sensory plasticity may involve catecholaminergic innervation of sensory neurons in the dorsal root ganglia (DRG).

Modulation of motor and sensory pathways of the peristaltic reflex by cannabinoids.

Cannabinoids have long been known to be potent inhibitors of intestinal and colonic propulsion. This effect has generally been attributed to their ability to prejunctionally inhibit release of acetylcholine from excitatory motor neurons that mediate, in part, the ascending contraction phase of the peristaltic reflex. In the present study we examined the effect of cannabinoids on the other transmitters known to participate in the peristaltic reflex using a three-compartment preparation of rat colon that allows separation of ascending contraction, descending relaxation, and the sensory components of the reflex. On addition to the orad motor compartment, anandamide decreased and AM-251, a CB-1 antagonist, increased ascending contraction and the concomitant substance P (SP) release. Similarly, on addition to the caudad motor compartment, anandamide decreased and AM-251 increased descending relaxation and the concomitant vasoactive intestinal peptide (VIP) release. On addition to the central sensory compartment, anandamide decreased and AM-251 increased both ascending contraction and SP release orad, and descending relaxation and VIP release caudad. This suggested a role for CB-1 receptors in modulation of sensory transmission that was confirmed by the demonstration that central addition of anandamide decreased and AM-251 increased release of the sensory transmitter, calcitonin gene-related peptide (CGRP). We conclude that the potent antipropulsive effect of cannabinoids is the result of inhibition of both excitatory cholinergic/tachykininergic and inhibitory VIPergic motor neurons that mediate ascending contraction and descending relaxation, respectively, as well as inhibition of the intrinsic sensory CGRP-containing neurons that initiate the peristaltic reflex underlying propulsive motility.