Gabapentin reduces painful bladder hypersensitivity in rats with lipopolysaccharide‐induced chronic cystitis

Abstract Although interstitial cystitis/bladder pain syndrome (IC/BPS) is a chronic condition causing bladder pain and urinary symptoms, effective treatments have not been established. The aim of this study was to adapt a chronic cystitis model in rats using lipopolysaccharide (LPS), which reflects IC/BPS pathology, and characterize the model's histological and behavioral effects. Furthermore, we investigated the effect of an α2δ subunit ligand, gabapentin (GBP), on bladder hypersensitivity of rats with chronic cystitis. Cystitis models were created by repeated intravesical injections of LPS. In the histological examination, the LPS‐injected group had greater inflammatory response, fibrosis, and abnormally thick re‐epithelialization. In the LPS‐injected group, LPS prompted hyperalgesia in both the lower abdomen and hind paw regions after day 1 of the first injection compared with the saline‐injected controls, without any recovery for 21 days at least. During cystometry, the LPS‐injected group showed bladder hyperactivity at all times. Systemic administration of GBP reduced cystitis‐related pain due to chronic inflammation and reduced the increased frequency of voiding in the LPS‐injected group. These results suggest that repeated intravesical injections of LPS induce long‐lasting bladder inflammation, pain, and overactivity in rats, while GBP is effective in the management of those symptoms in this chronic cystitis model. The current study identifies a relatively simple method to develop an animal model for chronic cystitis and provides evidence that GBP may be an effective treatment option for patients with IC/BPS.


| INTRODUC TI ON
Interstitial cystitis/bladder pain syndrome (IC/BPS) is a chronic bladder inflammation characterized by bladder and pelvic pain, and urinary symptoms, such as urinary frequency, urgency, and nocturia. 1,2 IC/BPS primarily affects women, but it can also occur in men over a broad age range, and severely affect the patient's quality of life. 3,4 The etiology of IC/BPS is still not completely understood, and effective drug treatments have not been established. 2 Although IC/BPS models have been evaluated by a variety of methods, [5][6][7][8][9][10][11] many of these are acute inflammation models. The durations of cystitis-related pain and bladder overactivity in these models are short, which are not consistent with the pathophysiology of chronic conditions displayed in patients with IC/BPS. Therefore, it is necessary to confirm the efficacy and safety of new therapies using an appropriate animal model with characteristics similar to those of the condition in human beings.
Lipopolysaccharide (LPS) is the main outer-membrane component of the gram-negative bacteria, including Escherichia coli, and acts as a common virulence factor. 12 Intravesical instillation of LPS induces an inflammatory response mediated by the activation of mast cells, production of cytokines, and recruitment of leukocytes to the mucosal surface of the bladder, similar to that observed in patients with IC/BPS. [13][14][15] Although repeated intravesical injections of LPS have reportedly increased the expression of macrophage migration inhibitory factor (a pro-inflammatory cytokine) in both the bladder and the lumbosacral spinal cord, 14 symptoms of bladder pain and bladder overactivity have not been demonstrated.
Gabapentin (GBP), an antiepileptic drug, has been effectively used in various chronic pain treatments and is especially suitable for neuropathic pain. [16][17][18][19][20] We and others have previously demonstrated that GBP acts as a neuromodulator by selectively binding to the α 2 δ subunits of voltage-gated Ca 2+ channels in various regions throughout the central nervous system, such as the locus coeruleus (LC) within the brainstem, and the spinal dorsal horn in rats after peripheral nerve injury. 17,21-23 As a result, it demonstrates a therapeutic effect on chronic pain by inhibiting the primary afferent traffic and excitation of the spinal nociceptive neurons. 24 Additionally, GBP has been observed to have a beneficial effect on bladder pain and overactivity related to IC/BPS. 25,26 In rodent acute cystitis models, a recent study reported that GBP could reduce detrusor overactivity and the visceral nociception, 27 but another study had failed to identify any effect. 28 Therefore, it is necessary to evaluate the effectiveness of GBP in chronic cystitis models with IC/BPS symptoms.
The current study confirmed whether repeated intravesical injections of LPS cause sustained bladder pain-related behavior and bladder overactivity in rats. Thereafter, we tested the effects of GBP on those symptoms in a rat model of LPS-induced chronic cystitis.

| Animals
Adult female Sprague-Dawley rats (Japan SLC, Hamamatsu, Japan), weighing 200-300 g, were used in this study. The animals were housed in a room maintained at 22-24℃ and 50%-60% relative humidity with an alternating 12-h light-dark cycle. Food and water were available ad libitum. All animal procedures were approved by the Committee of Animal Experiments, Tohoku Medical and Pharmaceutical University and were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals.

| Induction of LPS-induced cystitis
Rats were anesthetized with 2% isoflurane (Pfizer Inc). A PE-50 polyethylene tube (Becton Dickinson) was inserted into the bladder via the urethra to empty the bladder, and 0.5 mL of LPS (E. coli O55:B5, Sigma-Aldrich) at 1 mg/mL in sterilized saline was infused intravesically and remained in the bladder for 30 min. After the LPS exposure period, the bladder was rinsed once with saline and allowed to drain

| Histology
The bladders were fixed in 4% paraformaldehyde at 7, 14, and 21 days after the first LPS injection. The bladders of the control rats were collected 7 days after the first saline injection. Tissues were frozen and cut on a cryostat at 5-μm thickness, and then stained with hematoxylin and eosin (H&E) and Masson's trichrome staining for the histology of inflammatory cell infiltration and fibrosis, respectively. Gross histologic observations were performed using a microscope system (BZ-X800; Keyence).

| Drugs and administration
For oral administration, GBP (Tokyo Chemical Industry) was dissolved in distilled water and administered using a feeding tube (30-300 mg/6 mL per kg). For intravenous administration, GBP was dissolved in sterilized saline and injected (10-300 mg/3 mL per kg) at the rate of 1.2 mL/h.

| Behavioral studies
Hypersensitivity response to the lower abdomen and hind paw was assessed using calibrated von Frey filaments (Danmic Global, LLC). Rats were placed individually in a small acrylic cage with a wire mesh floor and acclimated to the experimental environment for 1 hour. In the abdominal stimulation, eight von Frey filaments exerting from 2 g to 60 g were used to assess the pain threshold. Tactile sensitivity of the region between the anus and urethral opening was assessed by applying the filaments perpendicularly to the surface of the skin. To prevent the wind-up effects of desensitization, repeated stimulation of the same location was avoided. Behaviors considered as positive response to filament stimulation were sharp retraction of the abdomen, immediate licking or scratching of the area of filament stimulation, and jumping.
For the hind paw stimulation, eight von Frey filaments exerting 0.6-26 g were used to assess the pain threshold. Filaments were applied to the plantar surface of the hind paw, and a brisk paw withdrawal was considered as the positive response. Withdrawal threshold was determined using an up-down statistical method. 29

| Cystometric studies
Cystometry was performed as we had reported previously. 30 Rats were anesthetized using 2% isoflurane, and a midline abdominal incision was made to expose the bladder. A PE-50 polyethylene tube with a fire-flared tip was implanted into the bladder dome for bladder filling and pressure recording 2 days before the experiments. A PE-10 polyethylene tube (Natsume Seisakusho) was inserted into the right jugular vein for intravenous drug administration. After surgery, rats were placed in a Ballman restraining cage (Natsume Seisakusho) and were allowed to recover from anesthesia for 1 hour. Physiological saline was infused at room temperature (22-24℃) into the bladder at a rate of 2.4 mL/h. Intravesical pressure was recorded using a force transducer, quad bridge amplifier FE224 (ADInstruments), and PowerLab data-acquisition system with LabChart Pro (ADInstruments). During the course of saline infusion, before drug administration, three voiding cycles were recorded as the control values, and each parameter was averaged.

| Statistics
Data were presented as the means ± SEM. Differences in the withdrawal threshold between the groups were analyzed using one-or two-way analysis of variance followed by either the Dunnett's test or Bonferroni test. The commercial software GraphPad Prism version 7 (GraphPad Software) was used to calculate statistical significance. P < .05 was considered significant. A parametric test (unpaired ttest) was used to test for differences in the cystometric variables between the two groups.

| Nomenclature of targets and ligands
Key protein targets and ligands in this article are hyperlinked to corresponding entries in http://www.guide topha rmaco logy. with saline-injected rats (control group) ( Figure 1A). Masson's trichrome staining in the LPS-injected group showed an increase in bladder tissue fibrosis, indicated by the blue coloring, compared with the control group ( Figure 1B).

| LPS-induced bladder pain-related behavior
At first, chronic cystitis due to repeated intravesical exposures to LPS was observed to induce persistent pain in rats ( Figure 2). Compared with saline-injected rats, the withdrawal thresholds in the abdomen ( Figure 2A) and the hind paw ( Figure 2B) were significantly reduced beginning at day 1 following the first injection of LPS and persisted until day 21 at least. The difference in body weight between the two groups was not significant (data not shown).

| LPS-induced increased bladder excitability
Representative traces of the cystometrograms obtained after repeated intravesical instillation of saline and LPS are shown in Figure 3A and B, respectively. In the control group, during saline  Figure 3C). The MVP of the LPS-injected group was slightly lower than that of the control group, although this was not significantly different ( Figure 3D).

| Effect of GBP on bladder pain-related behavior in LPS-induced cystitis
The analgesic effect of GBP on cystitis-related mechanical hyperalgesic behavior was tested at 7 days after the first LPS injection.
Orally administered GBP (30-300 mg/kg) suppressed mechanical hyperalgesia in the abdomen and the hind paw, evoked by LPS, in a dose-dependent manner (Figure 4). In the abdomen ( Figure 4A), oral GBP showed significant analgesic effects in doses of 100 and 300 mg/kg compared with that of the vehicle. In the hind paw ( Figure 4B), oral GBP showed significant analgesic effects from 30 to 300 mg/kg compared with that of the vehicle. The peak effect of oral GBP was observed 2-4 h after administration.

| Effect of GBP on bladder overactivity in LPSinduced cystitis
To test the therapeutic effect of GBP on overactive bladder symptoms, the effect of GBP on bladder overactivity was examined 7 days after the first LPS injection. In both the control and LPSinjected groups, intravenous administration of GBP (10-300 mg/kg) caused a dose-dependent prolongation of ICI (Figures 5-7A) without changing the MVP (Figures 5-7B). In the control group, intravenously administered GBP significantly prolonged ICI at a dose of 300 mg/ kg compared with that of the vehicle (P = .0045, n = 5, Figure 7A).
In the LPS-injected group, intravenously administered GBP significantly prolonged ICI in doses of 30 and 100 mg/kg compared with that of the vehicle (P = .0271, n = 6 and P = .0005, n = 6, respectively; Figure 7A). The prolongation of ICI due to GBP (30 and 100 mg/kg) in the LPS-injected group was significantly higher than that of the control group (P = .0156 and P = .0161, respectively; Figure 7A). In both groups, intravenous administration of GBP did not affect the MVP ( Figure 7B).

| DISCUSS ION
The clinical symptoms related to IC/BPS include urinary frequency, urgency, nocturia, and pain in the lower abdomen and pelvic regions; however, its response to current therapies and drugs is poor. 2

IC/
BPS is one of the refractory syndromes and its evaluation in animal models that can imitate the clinical aspects of the syndrome in humans is essential for therapeutic development. Therefore, we created a chronic cystitis model in rats, which demonstrated a long-lasting bladder inflammation, pain, and overactivity via repeated intravesical instillation of LPS. Additionally, the current study demonstrated that GBP, a drug used to treat chronic neuropathic pain, reduced bladder pain-related behavior and overactivity in this chronic cystitis rat model.

Cyclophosphamide (CPA) is most commonly used in rodents
for developing an experimental acute and/or chronic model for IC/ BPS. 6,8,9,33,34 However, CPA-induced chronic models lead to deleterious effects, such as strong body weight loss associated with high mortality. 35,36 Adjusting CPA doses can reduce weight loss and mortality but CPA-induced visceral pain does not appear sustained over long-term CPA induction. 36  GBP has been used as an effective analgesic for neuropathic pain, and its mechanisms of analgesic effect have been extensively studied. The current study demonstrated that GBP not only reduced bladder pain, one of the refractory pains, but also markedly reduced bladder overactivity in LPS-induced chronic cystitis rats.
Accordingly, GBP has been shown to be useful in overactive bladder as well as in bladder pain. GBP has a high affinity for the α 2 δ subunit of the voltage-gated Ca 2+ channels (particularly the N-type and L-type), 21,44 and specific binding to this subunit is crucial for analgesic effects. 17,45 As spinal plasticity and sensitization, including upregulated α 2 δ subunits, play pivotal roles in pain amplification after tivity of the bladder. 48 In the current study, GBP inhibited bladder overactivity in LPS-induced cystitis rats, but its inhibitory effect was poor in the control rats. This is consistent with previous reports that suggested a lack of effect in the absence of hypersensitivity. 16,18 This has been emphasized by a study that demonstrated GBP to specifically inhibit Ca 2+ currents in transgenic mice that overexpressed the α 2 δ-1 subunits, but did not affect the wild-type mice. 19 Furthermore, GBP may produce pain attenuating effects by acting on the supraspinal regions to stimulate bulbospinal descending inhibition and alleviate neuropathic pain. 18,20 GBP, both systemically administered in vivo and locally applied to isolated brainstem slices in vitro, activated noradrenergic neurons in the LC. 49 We and others have previously demonstrated that GBP reduces GABAergic activity in the LC by an interaction with the α 2 δ subunits, thereby increasing the activation of the descending pain inhibitory pathway to the spinal cord. 22,23 Interestingly, however, some α 2 δ subunit ligands, including 3-exo-aminobicyclo [2.2.1] heptane-2-exo-carboxylic acid (ABHCA), fail to produce behavioral analgesia, indicating the involvement of additional mechanisms. 19 We have previously demonstrated that GBP activates glutamate transporters and thereby facilitates glutamate-induced glutamate release in cultured astrocytes, while ABHCA do not. 50 An in vivo study also showed that GBP increased extracellular glutamate in the LC by astroglial glutamate transporter-mediated mechanisms to stimulate descending inhibition aside from α 2 δ subunits in neurons. 51 Thus, the antinociceptive effect of GBP is not solely responsible for the α 2 δ subunit, and the mechanism of action has not yet been definitively established. In the micturition reflex, Barrington's nucleus (Bar; also known as the pontine micturition center), located rostral and ventromedial to the LC, has been identified as the major brain center regulating urination. [52][53][54] The majority of neurons in Bar express a reporter for corticotropin-releasing hormone (CRH) 55-58 co-innervate spinal preganglionic neurons that control the bladder, 59,60 and LC neurons that provide noradrenaline innervation throughout the brain. 60,61 Furthermore, at least five separate population of neurons, including GABAergic and LC neurons, are located around Bar neurons. 57 Since increased CRH expression in and around Bar has been reported to prolong the ICI in rodents, [62][63][64] increased CRH may have an inhibitory effect on the micturition reflex. 65

D I SCLOS U R E
The authors have no conflict of interest to declare.

AUTH O R S CO NTR I B UTI O N S
Participated in research design: Yoshizumi and Mizoguchi.
Wrote or contributed to the writing of the manuscript: Yoshizumi, Watanabe, and Mizoguchi.

DATA AVA I L A B I L I T Y S TAT E M E N T
Additional information and requests for data should be directed to the corresponding author, Masaru Yoshizumi. Please contact yoshi-zumi@tohoku-mpu.ac.jp.