Presentation Authors: Taesoo Choi, Sung Tae Cho, Koo Han Yoo, Dong-Gi Lee*, Gyeong Eun Min, Seung Hyun Jeon, Hyung-Lae Lee, Seoul, Korea, Republic of
Introduction: Bladder stimulation induces up-regulation of neurotrophins which may contribute to voiding reflex. Elevated levels of neurotrophins have been detected in the urine and urothelium of individuals with interstitial cystitis. Phlogogenic bacterial infection involving the bladder can also be a stimulant to activation of its system resulting in pathological state. Phosphorylated responsive element of binding protein (p-CREB) is an important transcriptional factor in the neurotrophin signaling pathway. In vitro studies demonstrated that activation of downstream intracellular signaling molecules, especially transcription factors including CREB are important steps in neurotrophin-signaling cascades. One study reported that p-CREB was up-regulated in afferent neuron of rat DRG (dorsal root ganglia) by chemical induced cystitis. The aim of our study was to examine the change of p-CREB in rat DRG after repeated uropathogenic Escherichia coli (UPEC) infection of rat bladder.
Methods: The involvement of CREB signaling in acute and chronic E.coli infection was characterized by measuring p-CREB using a specific antibody. Adult female Spragueâ€“Dawley rats weighing 280 Â± 20g were prepared in this study. Total 19 rats were induced into acute E.coli infection (n=7) or into chronic E.coli infection (n=6) or control (n=6). After control or E.coli infection, all animals were anesthetized with sodium pentobarbital (50mg/kg, intraperitoneal) and then perfused with 0.05M phosphate-buffered saline (PBS), followed by 4% paraformaldehyde. After perfusion, the spinal cord and DRG were quickly removed and post-fixed for 6 hours. In DRG from control and acute/chronic cystitis rats, p-CREB cell profiles were counted in 6-10 sections of each DRG. For p-CREB immunoreactivity, DRG cells exhibiting intense nuclear staining were considered positively stained. The cell profiles of p-CREB immunoreactivity in each DRG section were presented as mean Â± standard deviations (SD) of the mean. All data were assessed using an ANOVA test for comparison among groups.
Results: In all groups, p-CREB immunoreactivity was observed in relatively small-diameter cell profiles with nuclear staining or nuclear & cytoplasmic staining in the DRGs (L1â€“L6, S1). p-CREB-IR in acute cystitis group did not show significant difference when compared with group A (p>0.05). In chronic cystitis group, p-CREB-IR in the L1â€“L6 and S1 DRG was significantly greater than control (p < 0.05), and p-CREB-IR in the L3â€“L6 and S1 DRG was significantly greater than that in acute cystitis group (p < 0.05). In control and acute cystitis group, p-CREB-IR in the L4-L5 DRG was significantly smaller than the other DRGs (p < 0.05). p-CREB-IR in the L6 and S1 DRG was significantly greater than L4-L5 DRG among chronic cystitis group (p < 0.05).
Conclusions: We observed the changes in the immunoreactivity of p-CREB in lumbosacral DRG cells after acute (single) or chronic (once a week for 4 weeks) E.coli cystitis. Especially, chronic E.coli infection up-regulated p-CREB expression in L1â€“L6 and S1 DRG, which means that the involvement of bladder afferent neurons cause its pathologic change. Under repetitive infection, p-CREB expression in DRG cells may be related to changes in bladder-originated factors influencing on micturition reflex pathways.