The Glt1 glutamate receptor mediates the establishment and perpetuation of chronic visceral pain in an animal model of stress-induced bladder hyperalgesia

Abstract

Psychological stress exacerbates interstitial cystitis/bladder pain syndrome (IC/BPS), a lower urinary tract pain disorder characterized by increased urinary frequency and bladder pain. Glutamate (Glu) is the primary excitatory neurotransmitter modulating nociceptive networks. Glt1, an astrocytic transporter responsible for Glu clearance, is critical in pain signaling termination. We sought to examine the role of Glt1 in stress-induced bladder hyperalgesia and urinary frequency. In a model of stress-induced bladder hyperalgesia with high construct validity to human IC/BPS, female Wistar-Kyoto (WKY) rats were subjected to 10-day water avoidance stress (WAS). Referred hyperalgesia and tactile allodynia were assessed after WAS with von Frey filaments. After behavioral testing, we assessed Glt1 expression in the spinal cord by immunoblotting. We also examined the influence of dihydrokainate (DHK) and ceftriaxone (CTX), which downregulate and upregulate Glt1, respectively, on pain development. Rats exposed to WAS demonstrated increased voiding frequency, increased colonic motility, anxiety-like behaviors, and enhanced visceral hyperalgesia and tactile allodynia. This behavioral phenotype correlated with decreases in spinal Glt1 expression. Exogenous Glt1 downregulation by DHK resulted in hyperalgesia similar to that following WAS. Exogenous Glt1 upregulation via intraperitoneal CTX injection inhibited the development of and reversed preexisting pain and voiding dysfunction induced by WAS. Repeated psychological stress results in voiding dysfunction and hyperalgesia that correlate with altered central nervous system glutamate processing. Manipulation of Glu handling altered the allodynia developing after psychological stress, implicating Glu neurotransmission in the pathophysiology of bladder hyperalgesia in the WAS model of IC/BPS.

Keywords: bladder pain syndrome; ceftriaxone; glutamate processing; hyperalgesia; interstitial cystitis.

Fig. 1.

Downregulation of an astrocytic transporter responsible for glutamate clearance (Glt1) during water avoidance stress (WAS). A: protein samples isolated from sacral spinal cord homogenates from Wistar-Kyoto (WKY) rats subjected to the specified days of WAS were analyzed by immunoblotting with antibodies specific for Glt1 (top). The relative protein levels were quantitated by densitometry and normalized to actin levels for consistency (bottom), demonstrating the downregulation of Glt1 levels in the spinal cord with increasing duration of psychological stress. Spinal samples were also examined after intraperitoneal administration of 200 mg ceftriaxone (CTX), which exhibited enhanced Glt1 expression. 3T3, lysates from NIH/3T3 cells, which do not express Glt1, served as a negative control. *P = 0.016. **P = 0.007. B: percent responses to stimulation of the bladder (visceral hyperalgesia) and the hindpaw (tactile allodynia) with an 8-g von Frey filament at the time points specified demonstrate increasing pain responses with greater duration of WAS. A minimum of 4 animals/treatment group were examined (n = 8 for t = 0, 10). NS, not significant. *P < 0.0001.

Fig. 2.

Voiding dysfunction after WAS. Following WAS, the average voided volume decreased significantly (A; P = 0.002), concomitant with increases in the number of voids (B; P = 0.03) compared with controls. C: we examined the ability of CTX to reverse WAS-associated voiding dysfunction. As seen in B, WAS increased the average number of voids per session compared with naive (Pre-WAS) or control animals. Intraperitoneal injection of CTX during WAS (WAS+CTX) decreased voiding frequency to baseline levels, while normal saline (WAS+vehicle) had no effect. When administered after the WAS period was completed (WAS+postCTX), CTX was still able to reverse the increases in voiding frequency seen after WAS, but this effect did not endure after CTX treatment was discontinued (WAS/postCTX Washout). Four animals per treatment group were examined. *P = 0.03. **P = 0.003. ***P = 0.001.

Fig. 3.

Dihydrokainate (DHK) administration induces visceral and tactile allodynia similar to WAS. A: percent withdrawal responses to suprapubic stimulation with von Frey filaments increased significantly after 10 days of WAS (visceral hyperalgesia). B: thresholds for withdrawal after hindpaw presentation (tactile allodynia) were significantly lower after WAS. A single intrathecal dose of DHK generated a pain phenotype indistinguishable from animals after WAS. Four animals per treatment group were examined. *P <0.001.

Fig. 4.

DHK does not alter voiding frequency. Control animals and animals subjected to WAS underwent intrathecal administration of vehicle or DHK before assessment of voiding frequency. While WAS treatment increased the average number of voids per 2-h period compared with control animals, DHK treatment did not alter the voiding frequency compared with similar animals treated with vehicle. Four animals per treatment group were examined. *P = 0.011. **P = 0.023.

Fig. 5.

CTX reverses induction of pain seen with WAS. A: while WAS was sufficient to significantly increase pain responses to suprapubic stimulation with von Frey filaments, administration of CTX during the stress period restored pain responses to baseline levels (WAS+CTX). CTX had no effect on animals who were not stressed (Control+CTX). B: decreases in withdrawal thresholds demonstrating tactile allodynia seen after WAS were abrogated by concurrent treatment with CTX during WAS. The key for B is the same as in A. Four animals per treatment group were examined. *P < 0.001.

Fig. 6.

CTX has no effect on pain parameters in naive animals before WAS. Animals were injected with vehicle alone or CTX before any handling or stressors. Regardless of CTX treatment (P = 0.972), the percent withdrawal responses to suprapubic stimulation with von Frey filaments (visceral hyperalgesia; A) were minimal. The thresholds for withdrawal after hindpaw presentation (tactile allodynia; B) were greater than the stimuli tested and unchanged by CTX treatment (P = 1). The key for B is as specified in A. Four animals per treatment group were examined. *P = 0.037.

Fig. 7.

Cephalothin (CLT) does not alter Glt1 levels in the spinal cord. A: protein samples isolated from sacral spinal cord homogenates from control rats and rats subjected to WAS were analyzed by immunoblotting with antibodies specific for Glt1 (top) and actin (bottom). B: Glt1 protein levels were quantitated by densitometry and normalized to actin levels for consistency. Again, CTX was observed to increase Glt1 levels over baseline. Also, WAS was again seen to downegulate Glt1 levels from control animals. CLT treatment, however, had no effect on Glt1 levels in either control animals or those subjected to WAS. Four animals per treatment group were examined. 3T3, lysates from NIH/3T3 cells, which do not express Glt1, served as a negative control. *P = 0.03. **P = 0.007.

Fig. 8.

CTX, but not CLT, reverses induction of pain by WAS. A: as seen in Fig. 5, treatment of rats with CTX during WAS (WAS+CTX) reversed the induction of higher pain responses seen after suprapubic stimulation with von Frey filaments, while treatment with CLT, a similar antibiotic without activity against Glt1, had no effect (WAS+CLT). B: a similar inhibition of tactile allodynia was seen in hindpaw stimulation with von Frey filaments following WAS with concurrent CTX, but not CLT, treatment. The key for B is the same as in A. Four animals per treatment group were examined. *P < 0.001. **P = 0.03.

Fig. 9.

Intrathecal DHK abrogates the inhibitory effect of CTX on pain induction during WAS. A: WKY rats were subjected to WAS and treated with CTX through the stress period. Previously placed intrathecal pumps delivered either vehicle or DHK continuously throughout the stress period. Animals receiving vehicle alone exhibited minimal pain to either suprapubic (A) or hindpaw (B) stimulation, due to the inhibitory effect of CTX on stress-induced pain. DHK infusion, however, abrogated the effect of CTX, resulting in elevated visceral hyperalgesia (A) and tactile allodynia (B). The key for B is the same as in A. Four animals per treatment group were examined. *P < 0.001.

Fig. 10.

CTX inhibits previously established pain. A: animals demonstrated enhanced visceral pain responses after completing the WAS protocol compared with controls (P < 0.001). Animals with established pain after completing WAS were treated with CTX for 5 days, which restored pain thresholds to control levels (WAS+pCTX); however, this pain inhibition did not endure after discontinuation of CTX treatment (WAS Washout). B: hindpaw stimulation was assessed in all animals before WAS or in control conditions; all animals exhibited equivalent, high pain thresholds (Pre-WAS). After 10 days of WAS (Was and WAS/pCTX), animals demonstrated significantly lower pain thresholds compared with control animals (Control and Control+pCTX). CTX administration after cessation of the inciting stress (Treatment) reversed the induction of pain in animals subjected to WAS (WAS+pCTX), but did not affect Control animals (Control+pCTX). The effect of CTX treatment to decrease pain responses, however, did not endure after removal of CTX (Washout). After discontinuation of CTX, all animals who had undergone WAS (WAS and WAS+pCTX) exhibited equivalent low pain thresholds, regardless of prior CTX administration. Four animals per treatment group were examined. *P < 0.001.

Fig. 11.

CTX inhibits previously established pain, even long after WAS. A: visceral pain responses 60 days after the initial testing period continued to be elevated in animals subjected to WAS (WAS d60) compared with control animals (Control d60). This increased visceral hyperalgesia could be effectively treated with CTX even at this late time point, restoring pain thresholds to control levels (WAS d60 +CTX). B: hindpaw stimulation demonstrated significantly lower pain thresholds in animals even 60 days after WAS (groups as designated in A). CTX administration 60 days after cessation of the inciting stress (WAS d60 +CTX) reversed this pain to levels similar to those seen in control animals. Four animals per treatment group were examined. *P = 0.002. **P < 0.001. ***P = 0.008.