The present study demonstrates that colonic inflammation increases the expression level of TRPV1 in the distal colon but not in the urinary bladder. Colonic inflammation-induced BDNF is expressed in TRPV1 nociceptive neurons and is attenuated by blockade of primary afferent activity with prolonged pre-treatment with RTX. Neutralization of BDNF in the DRG/spinal cord with intrathecal infusion of a BDNF antibody reverses bladder overactivity during colonic inflammation. These results suggest a role of primary afferent -mediated BDNF up-regulation in viscero-visceral cross-organ sensitization.
BDNF is a member of the neurotrophin family of growth factors consisting of nerve growth factor (NGF), neurotrophin-3 (NT-3) and NT-4. It has long been implied that BDNF plays a significant role in neuronal plasticity especially the long-term potentiation (LTP) of the central nervous system . Recent studies have also suggested a role of BDNF in modulating sensory activity in the peripheral nervous system. After peripheral inflammation, BDNF is synthesized in the primary sensory neurons in DRG where it facilitates intracellular signal transduction and gene expression at the dorsal horn of the spinal cord via anterograde transport [37, 43, 44]. As demonstrated in the present study, BDNF in the DRG is mainly expressed in TRPV1 nociceptive neurons. Blockade of primary afferents with the neurotoxin RTX reverses the BDNF up-regulation in the DRG during colonic inflammation, suggesting that signaling arising from the inflamed distal colon facilitates BDNF expression in the sensory neurons in DRG. Studies by us and others have demonstrated that colonic inflammation increases the level of NGF and/or neural activity in the inflamed colon [34, 45, 46]. NGF receptor TrkA is able to retrogradely transport from the inflamed colon to the DRG , where it may activate intracellular signaling molecules and regulate neuronal plasticity. The role of NGF/TrkA in regulating BDNF expression in the DRG has also been illustrated by previous studies showing that NGF treatment increases BDNF expression in the TrkA/CGRP peptidergic DRG neurons and almost 90% of TrkA DRG neurons express BDNF .
In addition to changes in the neurochemical coding of the sensory neurons during colonic inflammation, one of the major physiological alterations accompanying colonic inflammation is the bladder hyperactivity [7, 11, 12]. Analysis of cystometrograms reveals that the average inter-micturition intervals in control animals are 220 seconds when we infuse the urinary bladder with a saline solution at a speed of 9 mL/h. This paradigm results in, by calculation, an average of 0.55 mL infusing volume per micturition cycle, which is close to the directly measured amount of solution voided (an average of 0.537 g per micturition cycle). These results indicate that there was no leakage of solution into the abdomen from the intravesicle catheter insertion site during saline infusion. During colonic inflammation, we have found that the inter-micturition intervals are decreased and the quantity of urine voided is also decreased suggesting bladder overactivity in these animals. The bladder overactivity in colonic inflamed animals has also been confirmed via a non-invasive procedure in which the urine is collected naturally onto an underneath filter paper directly from the unrestrained nonoperated animals (Additional file 1). Analysis of the urine spots on the filter paper reveals that the animals excrete fewer times (i.e., lower number of urine drops) with larger volumes per drop (i.e., bigger urine spots) before induction of colonic inflammation. TNBS-treated animals void more frequently (i.e., higher number of urine drops) with smaller quantities of urine per voiding (i.e., smaller urine spots) (Additional file 1). Both techniques used in the current study demonstrate that colonic inflammation indeed induces bladder hyperactivity with minimum change in the morphology of the urinary organ, suggesting a neurogenic mechanism in colon-to-bladder cross-sensitization. It is noteworthy that there are increased numbers of non-voiding contractions detected in the inflamed animals. These non-voiding contractions may be due to the hyperactivity of the urinary bladder per se, or due to the abdominal contraction pressure transference to the viscera in the inflamed animal. Further examination of the activity of the abdominal wall will identify the mechanisms.
The mechanisms and pathways that mediate colon-to-bladder cross-organ sensitization have been vigorously studied in the past years. A growing body of evidence suggests that activation of the primary afferent pathway and a neural cross talk or interaction in the DRG and spinal cord has a significant role in mediating cross-organ sensitization [12, 14–16, 18, 19]. The primary afferents that innervate the urinary bladder or the distal colon are located in the same spinal segments [19, 48, 49]. Thus, irritation of one visceral organ, such as the distal colon in the present study, may lead to activation of the primary afferent neurons projecting to this organ and cross-activation of the nearby afferent neurons projecting to a different viscus such as the urinary bladder. An experiment involving injection of a viral vector encoding NGF to the urinary bladder demonstrates that over-expression of NGF triggers the hypersensitivity of remote organs including the distal colon . During colonic inflammation, the level of NGF is significantly increased in the distal colon [34, 45]; the elevated NGF may have a role in triggering bladder hyperactivity by modulating the plasticity of sensory neurons. Modified bladder spinal reflex may regulate the detrusor muscle contractility and/or the activity of the urethral sphincter leading to increased voiding frequency.
BDNF elevated in the sensory neurons during colonic inflammation may modulate the bladder sensory activity. The level of BDNF high affinity receptor TrkB is increased in bladder afferent neurons during colonic inflammation . The accumulation of TrkB in bladder afferent neurons may enhance the responsiveness of these neurons to BDNF, thus leading to changes in plasticity of these neurons. In DRG neuron culture, BDNF increases the expression level of CGRP , an excitatory neurotransmitter that is also up-regulated in the bladder afferent neurons during colonic inflammation , suggesting a possible role of BDNF in modulating bladder afferent excitability.
The role of BDNF in regulating visceral sensitivity has been suggested by a previous study showing that intraperitoneal injection of BDNF neutralizing antibody attenuates colonic inflammation-induced colonic hypersensitivity . To specifically target the BDNF that is expressed in the DRG and spinal cord , in the present study a specific BDNF neutralizing antibody was infused into the DRG/spinal space under the dural membrane. This treatment regime also blocks colonic inflammation-induced bladder hyperactivity. BDNF that is up-regulated in the sensory neurons following colonic irritation may be released into the extracellular space within the DRG and the spinal cord. The action of BDNF is then blocked by the intrathecal BDNF antibody that further blocks the sensitivity of the bladder afferent neurons and modulates the urinary bladder function. Bladder afferent nerves consist of myelinated Aδ-fibers and unmyelinated C-fibers projecting to the lumbosacral spinal level which is also of major importance in controlling colonic function [50, 51]. Activation of the C-fibers during colonic inflammation is apparent from our studies showing that the level of TRPV1 is significantly increased in nerve fibers in the inflamed distal colon. Blockade of the activity of these nerves with prolonged pre-treatment with RTX has also blocked colonic inflammation-induced bladder overactivity.