Hypothesis / aims of study
Chronic stress plays a substantial role in the development, maintenance and enhancement of functional bladder disorders including painful bladder syndrome/interstitial cystitis (PBS/IC). Studies have shown that more than half of patients with PBS/IC report daily or constant pain and urinary frequency, which are exacerbated by stressful circumstances. In fact, patients tell their physicians that stress plays a major role in their symptom flares. However, the mechanisms underlying the relationship between stress and hypersensitivity of the urinary bladder are not well understood. Altered levels of neurotrophins have been correlated with bladder hyperexcitability and pain. Artemin is a glia derived neurotrophic factor that supports the survival and development of a group of primary sensory neurons. Preclinical models for chronic pain disorders as well as clinical trials in patients with chronic pain (NCT00961766, NCT01873404), revealed that artemin exhibits the ability to modulate pain responses. Thus, our overall hypothesis is that the levels of artemin and/or its receptor are altered in PBS/IC bladder, which leads to sensory afferent hyperexcitability, changes in nociceptive channel expression/function and resultant bladder pain. Modulating levels of artemin (using immunotherapy with neutralizing antibodies) could in turn, reduce afferent neuron excitability and bladder pain.
In this study, we examined the influence of psychological stress on artemin levels, bladder hyperalgesia and properties of the primary afferent neurons.
Study design, materials and methods
This study was conducted in an animal model consisting of a rat genetically predisposed to stress, Wistar Kyoto, exposed to psychological stress: water avoidance stress or WAS.
• Female Wistar Kyoto rats (~200g; 3-4 month old) were exposed to WAS for 1h per day for 1, 3, 6, or 10 days (versus handled groups as controls; n=6-12 rats per group). At the end of the stress paradigm rats were placed for 24h in metabolism cages to assess bladder function, followed by pain tests using von Frey filaments, and then sacrificed for tissue collection (blood, bladder, L6 and S1 dorsal root ganglia - DRG).
• To assess the role of artemin a group of rats were treated with anti-artemin antibody (100 µg/kg, i.p) daily throughout a 6day WAS protocol.
• Circulating artemin levels (plasma) were measured using ELISA. Artemin expression in bladder tissue was evaluated using western blot.
• Acutely dissociated DRG neurons were loaded with fura-2AM to measure intracellular calcium concentration ([Ca2+]i). As artemin supports sensory neurons that express TRPA1 channels, we focused on calcium responses to the TRPA1 agonist, mustard oil (MO, 100 µM).
a) WAS alters artemin levels in a time-dependent manner
Circulating plasma artemin and artemin expression in bladder tissue increased with ‘acute’ WAS (1-6 day) and significantly decreased with ‘chronic’ WAS (10 day). Treatment with anti-artemin antibodies during the acute phase (1-6d WAS) significantly decreased circulating plasma levels.
b) Chronic WAS alters bladder function
Evaluation of bladder function in awake rats using metabolism cages, revealed increased micturition frequency and decreased voided volume in the 10d WAS group (chronic stress). No changes were observed at earlier time points. Similarly, cystometry revealed ~25% decrease in intercontraction interval in the 10d WAS rats compared to controls.
c) WAS and artemin levels modulate visceral sensitivity
Von Frey assessment of visceral sensitivity indicated ~70% decrease in the threshold for visceral sensitivity in WAS rats compared to controls. This decrease was partially prevented by anti-artemin antibody treatment.
d) WAS and artemin levels modulate TRPA1 responsiveness in DRG neurons
DRG neurons from chronic WAS and anti-artemin treated WAS rats exhibited decreased responses to the TRPA1 agonist, mustard oil, compared to controls. DRG neurons exposed to acute in vitro artemin treatment (100ng/ml for 1h) exhibited increased responses to mustard oil.
In summary, alterations in artemin during stress (WAS) correlate with both visceral hypersensitivity and TRPA1-mediated afferent neuron sensitivity. WAS also alters bladder function resulting in symptoms similar to those experienced by PBS/IC patients (i.e. increased voiding frequency).
Interpretation of results
These findings indicate for the first time that artemin levels exhibit a pattern that correlates with the duration of the physiological stress. Artemin increases in early phases of stress (e.g. 1-6day WAS) and decreases with ‘chronic’ stress (10day WAS). We find that decreasing artemin levels partially mitigated visceral hyperalgesia which supports the view that an early increase in artemin could sensitize nociceptive afferents, contributing to acute pain. Because recent findings show that many IC patients are high stress responders, these findings may represent an underlying mechanism for exacerbation of IC pain during flares.
As artemin is a growth factor involved in synapse formation, maintenance and strengthening, higher levels of artemin may promote formation of aberrant synapses, that would conceivably impair sensory signalling and contribute to disrupted bladder function and/or pain. With transition from acute to chronic state, sensitized neurons may accumulate higher levels of calcium through activation of TRPA1 channels and may need survival support. If artemin levels decrease, that support is missing/diminished and thus these neurons are further pushed towards cellular disruption and possibly death. These mechanisms may account for chronic pain. Future studies are needed to examine these hypotheses.
Chronic stress triggers a number of changes that ultimately can exacerbate or predispose to disease such as PBS/IC. These novel findings support the concept that stress can alter levels of the neurotrophic factor artemin, which can impact the expression and/or function of nociceptive TRPA1 channels and contribute to visceral sensitivity and pain. Thus, fluctuations in artemin levels with progression of stress-induced cystitis may correlate with neuronal sensitization and aberrant signalling in the periphery and spinal cord. From a clinical perspective, these experiments provide important insights regarding an appropriate window for treatment. For example, intervening with anti-artemin treatment during the initial ‘acute’ state may prevent neuronal changes, but if this treatment is administered at a later stage it may be deleterious. At later chronic stages, increasing artemin levels and/or activating artemin intracellular pathways may be beneficial.
Manipulation of artemin expression and/or signalling pathways may therefore offer a new pain treatment strategy for PBS/IC patients.