Sex as a biological variable. This study employed female young (3 months) Harlan Sprague-Dawley rats from Envigo. The CYP-induced cystitis model is well established using female rats to examine inflammatory and nociceptive pathways. However, we do not rule out the possibility that our findings could be relevant to more than one sex, as CYP-induced HC has been reported to occur in both male and female patients.

Animals. Bladder cystitis was induced with 3 injections of 75 mg/kg (i.p.) of CYP (Cayman Chemical) given on days 0, 3, and 6. 8-AG (5 mg/kg/d, Toronto Research Chemicals) was administered in drinking water with daily dosing monitored. This dose of 8-AG was selected based on our preliminary studies showing that 5 mg/kg/d provides a urinary concentration of 8-AG approximately 15 ± 3 (mean ± SEM; n = 4) µmol/L, a concentration that is at least 5 times the inhibition constant, or KI, of 8-AG against PNPase (KI estimated by us to be 2.8 µmol/L against human recombinant PNPase with inosine as substrate) (72). Animals were randomly assigned into the following groups: 1) vehicle control, 2) CYP treatment only (sacrificed on day 8), and 3) treatment with both 8-AG and CYP, whereby 8-AG is started 14 days prior to the start of CYP. In a separate set of animals, 8-AG treatment was begun 24 hours after the start of CYP. All animals were sacrificed on day 8.

Voiding analysis. Control, CYP-treated, and 8-AG–treated rats were placed in metabolic cages on days 7–8 of the CYP dosing regimen. The light cycle was from 7 am to 7 pm, and food and water were provided ad libitum. Voided urine was captured and measured on a plate/load cell system controlled by a comprehensive lab animal monitoring system (Columbus Instruments). Data were analyzed on LabChart software (ADInstruments), averaged for 24 hours, and analyzed for 12-hour periods during the day (7 am—7 pm) and night (7 pm—7 am). Voiding frequency (voids per hour), intervoid interval, and volume per void were analyzed. Voiding frequency was calculated as the number of voiding events per hour during 24 hours and during the 12-hour day and 12-hour night periods. Volume per void, which defines bladder capacity, was calculated as an average of the voids occurring during these periods.

Von Frey testing. On day 7–8 of the CYP protocol, pain behaviors were assessed by scoring animals’ behaviors in response to application of von Frey filaments following a testing protocol developed by Auge et al. (18). Baseline testing was performed prior to any treatments (CYP or 8-AG). Testing was performed by a single experimenter. Rats were placed in individual transparent cages placed on top of an elevated mesh stand support (Bioseb) and were allowed to acclimate for a minimum of 30 minutes, until each rat was resting quietly. Increasing filaments within a set of 8 (1.4, 2, 4, 6, 8, 10, 15, and 26 g, Stoelting Co.) were each applied 3 times, with a minimum of 5 seconds between each test. Each instance was scored on a scale as follows: 0 = no response, 1 = reaction of the animal, 2 = reaction and change of position, or 3 = reaction, change of position, and licking and/or vocalization. Nociceptive score for each filament was calculated as a percentage of the maximal possible score.

Single unit afferent nerve recordings. Single unit afferent nerve activity was recorded from the sacral S1 dorsal root fibers of female Sprague-Dawley rats receiving no intervention or treatment with CYP or CYP with 8-AG pretreatment using a previously described methodology (73, 74). Briefly, the urinary bladder was dissected with associated S1 dorsal roots, and the bladder was cut along the ventral aspect to form a sheet. The bladder/nerve preparation was placed into a temperature-controlled organ bath perfused with oxygenated Krebs solution. The base of the bladder was secured with pins to the chamber, and the dome was connected to a tension transducer in line with a computer-controlled micromanipulator. The nerve roots were passed into bilateral oil chambers and split into 10 to 20 fine filaments. The filaments were wrapped around a platinum-iridium recording electrode to measure biphasic depolarization in response to mechanical distention of the bladder. Controlled stretches were applied to the bladder using a computer prompt, varying the distance, speed, and duration of the stretch protocol. The velocity of stretch ranged between 0.2 to 0.8 millimeters/second with a 30-second hold followed by a return to baseline. Recorded units were classified based on amplitude. Mechanosensitivity of individual units was determined by linear regression of total counts or counts/second plotted against mean tension during the 30-second hold.

Vascular alterations. On day 8 of the CYP protocol, animals were anesthetized by isoflurane, and the bladder was surgically exposed for assessment of vascular blood perfusion. Real-time blood perfusion (1 mm3 tissue) was accomplished using a BLF22D laser Doppler flowmeter (Transonic Systems, Inc) with a surface probe (TypeS-APLPHS) applied to the serosal surface of the bladder (apex and neck) wall using Doppler light shift from moving RBCs to analyze flow by the Bonner algorithm. This method gives robust, noninvasive microvascular flow signals in the bladder wall of anesthetized rats (29).

Histopathology. Following blood flow measurements, rats were sacrificed under isoflurane anesthesia. Gross macroscopic observations of bladder petechiae were noted and images taken with an Olympus SZX16 dissecting microscope using cellSens software (Olympus). Bladders were dissected and prepared for histopathology and molecular studies. For histological assessment of urinary bladder, sections of bladders were fixed flat in 10% formalin. After embedding in paraffin, sections were stained with hematoxylin and eosin by the UPMC Research Pathology Core shared resource. Images were collected on an Olympus BX-63 microscope using Olympus cellSens software and assessed by an independent pathologist for morphological changes in the urothelial mucosa.

Western immunoblotting. Bladder preparations (including both full wall thickness and mucosa separated from the underlying lamina propria and detrusor smooth muscle) were homogenized using Lysing Matrix D in a FastPrep 24 instrument (MP Biomedicals) in HBSS (5 mM KCl, 0.3 mM KH2PO4, 138 mM NaCl, 4 mM NaHCO3, 0.3 mM Na2HPO4, 5.6 mM glucose, and 10 mM HEPES, pH 7.4) containing complete protease inhibitor cocktail (1 tablet/10 mL, Roche) and phosphatase inhibitor cocktail (MilliporeSigma, 1:100). After centrifugation (16,200g; 15 minutes at 4°C), the membrane protein fraction was prepared by suspending the membrane pellets in lysis buffer containing 0.3 M NaCl, 50 mM Tris-HCl (pH 7.6), 0.5% Triton X-100, and the same concentration of protease inhibitors as above. The suspensions were incubated on ice and centrifuged (16,200g; 15 minutes at 4°C). The protein concentrations of the combined supernatants were determined using the Pierce BCA protein assay (Thermo Fisher Scientific). After denaturation (100°C for 5 minutes) in the presence of Laemmli sample buffer, lysate from each sample was separated on a 4%–15% TGX Stain-Free SDS-PAGE gel (Bio-Rad). As a reliable loading control, total protein measurement per sample was determined using Bio-Rad Stain-Free SDS-PAGE technology. Ultraviolet-activated protein fluorescence within the gel was imaged on a ChemiDoc MP (Bio-Rad). After proteins were transferred to PVDF membranes, total protein fluorescence of the membrane-bound proteins was imaged on a ChemiDoc MP (Bio-Rad), and the membranes were incubated in 5% (weight/volume; w/v) dried milk dissolved in TBS-T (20 mM Trizma, 137 mM NaCl, 0.1% Tween 20, pH 7.6), rinsed with TBS-T, and incubated overnight at 4°C with primary antibody: cytokeratin 20 (Abcam AB230524), HIF-1alpha (Abcam AB216842), Mst3b (Cell Signaling Technology 4062S), nitrotyrosine (Enzo Life Science BML-SA468-0100), NLRP3 (Abcam AB214185), PECAM (Novus Biologicals NB100-2284), PNPase (Atlas Antibodies HPA001625), TLR4 (Santa Cruz Biotechnology SC-293072), and UPIII (Abcam AB231576) diluted in TBS-T containing 5% (w/v) milk or 5% (w/v BSA). After washing in TBS-T, the membranes were incubated with secondary antibody (sheep anti-mouse HRP Cytiva, NA931-1ML; or donkey anti-rabbit HRP, Advansta, R-05788-500) for 1 hour in 5% (w/v) milk TBS-T, washed, incubated in WesternBright Quantum (Advansta), and imaged on a ChemiDoc MP (Bio-Rad). Data were quantified and analyzed in Image Lab software (Bio-Rad). The volume (intensity) of each protein species was determined and normalized to total protein imaging of the membrane using Bio-Rad Stain-Free SDS-PAGE technology.

Measurement of inosine in bladder and hypoxanthine in culture medium. Inosine in bladder and hypoxanthine in culture medium was measured by ultraperformance liquid chromatography-tandem mass spectrometry as recently described by us (75).

Microglial histology and activation. Under isoflurane anesthesia, rats were transcardially perfused with PBS containing 100 U of heparin, followed by 4% paraformaldehyde. SC segments (L6-S1) were sucrose-protected, frozen in optimal cutting temperature compound, and sectioned at 20 μm on a Leica cryostat onto slides. Sections were rinsed in PBS (2.68 mM KCl, 1.47mM KH2PO4, 137 mM NaCl, 8 mM Na2HPO4, pH 7.4) and exogenous peroxidase–blocked with 1% hydrogen peroxide and nonspecific staining blocked with 0.5% BSA, then incubated overnight at 4°C with rabbit anti–IBA-1 (Wako 019-19741). After rinsing with PBS, sections were incubated with biotinylated goat anti-rabbit secondary antibody and then avidin-biotin peroxidase (Vectastain ABC kit, Vector Laboratories), visualized with VIP peroxidase substrate (Vector Laboratories), and counterstained with methyl green. Sections were dehydrated through xylene and ethanol gradients before coverslipping with VectaMount permanent mounting medium (Vector Laboratories). Images were taken of the IBA-1–stained sections (×40 original magnification) and morphological characteristics of the stained microglia were analyzed using Olympus cellSens software. In the standard assessment of microglia, the degree of activation is graded based on a visual analysis of morphological properties as described by Kreutzberg (76). Microglia were both qualitatively assessed (e.g., activation exhibits both retraction of processes and increased cell body size) and quantitatively assessed using a modification of previously published methods (69). A minimum of 3 immunohistochemically labeled sections per SC region were analyzed by a researcher blinded for the treatment. Using ImageJ (National Institutes of Health), a standardized region of interest was selected, and the IBA-1–stained section was isolated into a single-color image followed by the threshold function to create a binary image. The total cell size, including the entire cell body plus projections, was measured by the analyze particle function with no size filter. Next, the microglial central cell body was selected, and the analyze particle function was used to measure the cell body size. The cell body–to–cell size ratio was then calculated and used as a quantitative measurement of microglial activation.

Mitochondrial respiration. Mitochondria were isolated from control, CYP-treated, and CYP-treated with 8-AG Sprague-Dawley rat SCs as described previously (77) with modifications. Briefly, after deep anesthesia with isoflurane (5%), the SC segments L6-S1 (≈80 mg) were isolated by extrusion (78) and placed in a mitochondrial solution containing 5 mM HEPES, 125 mM KCl, 2 mM Pi, 20 μM EDTA, 5 mM MgCl2, and 0.2 mg/mL BSA, adjusted to pH 7.4 with 100 mM KOH. The tissue was then minced by a McIlwain motorized tissue chopper (Brinkmann) set to chop at a 10 μm interval. The minced tissue was placed in 10 mL of mitochondrial solution and homogenized by a few passes with a motorized Teflon pestle. The homogenate was spun at 1,000g for 10 minutes. The supernatant was then spun at 10,000g for 10 minutes to obtain a second pellet containing the mitochondria. This pellet was resuspended in 100 μL of mitochondrial solution, and 25–50 μL of the suspension was placed in a gas-tight vessel containing a Clark-type oxygen microelectrode (MI-730/OM-4; Microelectrodes) to measure the state 3 (succinate + ADP) and state 4 (succinate alone) respiratory rates. The electrode was calibrated (79) considering a total amount of dissolved O2 in aqueous solution after equilibration with air at 36°C to be 215 μM, zeroed with sodium dithionite. The RCR, a measure of the “tightness of coupling” between electron transport and oxidative phosphorylation, was determined from the ratio of state 3 to state 4 rates of respiration. An RCR of 2–4 is considered acceptable for complex II substrates (80).

Neuronal cultures. Cortical neurons were harvested from E16–E18 Sprague-Dawley rat embryos as previously described (81, 82). Briefly, both male and female embryonic brains were isolated, trypsinized, triturated, and seeded on 10 cm poly-d-lysine culture plates (BD Biosciences). Neurons were grown in Neurobasal medium (Life Technologies) containing B27 supplement, 0.5 mM glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin. Neurons (95% pure) were maintained by one-half medium exchange 3 and 5 days after culture. Experiments were conducted on in vitro day 6 (DIV6).

Astrocyte cultures. Astrocytes were harvested using our previously published protocol (81, 83). Briefly, brains were isolated from male and female postnatal day 1–2 Sprague-Dawley rat pups. The tissue was isolated, trypsinized, triturated, and seeded onto 75 cm2 tissue culture flasks. Cells were grown to 90%–95% confluence in DMEM/F12/10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin. Experiments were conducted in 10 cm poly-d-lysine culture plates after several propagations to select astrocytes only.

Microglia cultures. Primary microglial cultures were isolated from postnatal day 1–2 male and female Sprague-Dawley rat pups using a modification of the method described by Ni and Aschner (81, 84). In brief, microglia cultures were harvested following the same protocol used to harvest astrocytes, except that the resulting brain cell mix was plated onto 225 cm2 flasks (about 4 to 6 brains per flask). Cells were plated and grown in DMEM/F12/10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin. The flasks were left untouched for the first week following culture, then given complete medium exchange every 2–3 days for 2 weeks. Three weeks after culture, cells were given a complete medium exchange, and flasks were shaken at low speed for 8 minutes, resulting in the dissociation of microglia cells from the flask into the media. The media were collected and centrifuged for 5 minutes at 200g and 4°C. Microglia were plated onto 35 mm poly-d-lysine culture plates at a density of 500,000 cells per plate. Experiments were performed 1 day after plating to avoid growth of astrocytes.

Validation of cell type enrichment in primary CNS cultures. DIV6 neurons, DIV1 microglia, and confluent monolayer astrocytes were harvested in RIPA buffer supplemented with EDTA and protease/phosphatase inhibitors. Cell types were characterized by Western immunoblotting using markers for neurons, astrocytes, and microglia as described previously (85).

RT-qPCR. Total RNA was isolated from neurons, astrocytes, and microglia using TRIzol reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions. The cDNA was synthesized using iScript cDNA synthesis kit (Bio-Rad). qPCR analysis was performed using Power SYBR Green PCR Master Mix (Thermo Fisher Scientific) in the Applied Biosystems QuantStudio 3 Real-Time PCR System (Thermo Fisher Scientific). Primers for PNPase were 5′-TGATCTGTGGTTCCGGCTTA-3′ (forward) and 5′-CACTGGGAACGTCACCTTG-3′ (reverse); for β-actin 5′-ACTCTTCCAGCCTTCCTTC-3′ (forward) and 5′-ATCTCCTTCTGCATCCTGTC-3′ (reverse). Threshold cycle (Ct) for β-actin was subtracted from Ct for PNPase to calculate 2ΔCt.

Statistics. Data were analyzed in GraphPad Prism 10 by ordinary 1-way ANOVA followed by either Tukey’s post hoc or Newman-Keuls post hoc multiple-comparison test. P < 0.05 was considered significant. Results are expressed as means ± SD.

Study approval. The Institutional Animal Care and Use Committee of the University of Pittsburgh approved all procedures. The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).

Data availability. Data are available in the paper’s supplemental material (Supporting Data Values) or from the corresponding author upon request.