The expression of NGF mRNA and protein was increased in a time-dependent manner (day 1 to day 7) in colonic SMC, but not in mucosa/submucosa of the obstructed colon. enhanced, and the withdrawal response to VFF stimulation markedly increased in BO rats. The expression of NGF mRNA and protein was increased in a time-dependent manner (day 1 to day 7) in colonic SMC, but not in mucosa/submucosa of the obstructed colon. Mechanical stretch caused robust NGF mRNA and protein expression in colonic SMC. Treatment with anti-NGF antibody attenuated colon neuron hyper-excitability and referred hypersensitivity in BO rats. Obstruction led to significant increases of tetrodotoxin-resistant (TTX-r) Na+ currents and mRNA expression of Nav1.8, but not Nav1.6 and Nav1.7 in colon neurons; these changes were abolished by anti-NGF treatment. In conclusion, mechanical stress-induced upregulation of NGF in colon SMC underlies the visceral BMS-740808 hypersensitivity in BO through increased gene expression and activity of BMS-740808 TTX-resistant Na+ channels in sensory neurons. Keywords: Lumen distension, Visceral sensitivity, Mechano-transcription, Dorsal root ganglia, Sensory neurons INTRODUCTION Obstructive bowel disorders (OBD) are characterized by lumen distention due to mechanical or functional obstruction in the gut. OBD represent a significant health challenge in adults and children [32, 45]. Mechanical bowel obstruction (BO) is one of the most common causes of acute abdomen [29, 32, 45], and accounts for 300,900 hospital admissions per year in the US alone; the aggregate cost for hospital stay is more than $2.7 billion annually, BMS-740808 topping all other gastrointestinal (GI) conditions [29]. Abdominal pain and motility dysfunction are the main complaints in BO [33, 36, 42]. While surgical resolution is the treatment of choice for many patients with mechanical BO, about 43% of patients receive nonsurgical conservative management [10, 46]. Abdominal pain is a major focus in conservative management, especially among those with inoperable or malignant obstruction [18, 34, 35]. Mechanical BO occurs in up to 25% of patients with colon cancer and 42% with ovarian cancer [33, 35]. High dose opioids BMS-740808 are the primary analgesic treatment for BO-associated pain in such cases [18, 33C35]. However, opioids are known to further cause opioid-induced bowel dysfunction, i.e. constipation and narcotic bowel syndrome [14, 19]. In addition, distention-associated abdominal pain and discomfort are major symptoms in chronic functional obstruction, such as intestinal pseudo-obstruction and Hirschsprungs disease [8, 16, 30]. The mechanisms of distension-associated abdominal pain in mechanical and functional obstructions remain unknown, and no specific analgesics are available to target distension-associated pain. Visceral sensitization is a well-recognized contributor to abdominal pain [1, 3, 12, 28]. The nerve endings of primary afferent neurons sense chemical and mechanical stimuli in the gut [5, 44] and transduce the signals to the neuronal cell body located in dorsal root ganglia (DRG), which relays the signals to the second order neurons in the spinal cord to initiate central processing of sensory information for perception [1, 3, 11, 12]. Several peripheral mediators including neurotrophins (NT), such as nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF), may sensitize afferent neurons [9, 31, 52]. This mechanism of peripheral sensitization is critical to the development of abdominal pain [1, 3, 9, 11, 31, 52]. Recent studies found that the firing threshold of colon-projecting afferent neurons and the pain threshold to abdominal stimulation were decreased in colon obstruction mice [17]. These data suggest that visceral sensitivity is increased in BO. However, what accounts for the increased visceral sensitivity in obstruction remains largely unknown. Mechanical distention is a cardinal feature in OBD [32, 36, 42, BMS-740808 45]. Mechanical stress was found CBL2 to increase NGF expression in vascular smooth muscle cells (SMC) [7]. We tested a hypothesis in the present study that lumen distention associated mechanical stress induces gene expression (mechano-transcription) [22, 25, 27, 39, 50] of NGF in colonic SMC, and that mechanical stress-induced NGF from SMC sensitizes afferent neurons and contributes to abdominal pain in OBD. We found that NGF expression in colonic smooth muscle was significantly up-regulated by mechanical stress in distended colon segment in a rat model of bowel obstruction and in the primary culture of rat colonic SMC study protocol as described previously [26] with modifications. In brief, muscle strips of 20 mg were isolated from the colon segment oral and aboral to BO (day 3), and from the sham controls. The strips were incubated in 1 mL of DMEM (+1% FBS) for 24 h, and the conditioned media were collected. Normal colon projecting DRG neurons were isolated from T13-L2 of na?ve rats,.
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