Neuroscience Capstone
Independent Research
Interstitial cystitis/bladder pain syndrome (IC/BPS) is a chronic pelvic pain condition characterized by bladder pain, urinary urgency, and frequency in the absence of overt infection or pathology. Despite its substantial impact on quality of life and its marked sex bias in diagnosis, the mechanisms driving IC/BPS pain and inflammation remain poorly defined, and current treatments are often only partially effective.
In this preclinical study, we used a VEGF-based mouse model of IC/BPS to investigate inflammasome signaling, pain and to test whether targeting this pathway with novel compounds can alleviate pain- and voiding-related outcomes.
Under the mentorship of Dr. Tyler Nguyen and Dr. Bethany Neal-Beliveau, I played a primary role in the behavioral assessments, including von Frey testing, Mouse Grimace Scale scoring, and void spot analysis, and contributed to experiments combining in vivo bioluminescent imaging and ex vivo tissue analysis to link caspase-1-dependent signaling with IC/BPS-like phenotypes. This site presents the project in an article-style scientific format, highlighting the experimental design, key findings, and limitations, while also showcasing my ability to communicate complex neuroimmune mechanisms clearly to different audiences.
Thank you for your time and interest in this work.
Abstract
Investigating the Role of NLRP3/Caspase-1-Related Inflammation in Interstitial Cystitis/Bladder Pain Syndrome (IC/BPS)
Interstitial cystitis/bladder pain syndrome (IC/BPS) affects many with chronic pelvic pain and urinary issues, with no known cause and limited treatment options. Elevated VEGF-A and IL-1β levels in IC suggest inflammation is key. Transurethral VEGF-A in rodents creates a reliable IC model. Using this alongside our inflammasome bioluminescence reporter mouse, we aim to study the role of NLRP3/caspase-1-driven inflammation in IC. We hypothesize that caspase-1 inflammation drives pathological changes, micturition problems, and pain behaviors in IC.
Caspase-1 reporter mice with thinned-skull cranial windows received three bladder instillations of VEGFA. They underwent in vivo bioluminescence imaging (IVIS) at baseline and 24 hours after each instillation to assess caspase-1 activation. Simultaneously, their micturition and pain behaviors were evaluated using Void Spot Assay, Abdominal Von Frey, and Grimace testing. Ex vivo IVIS imaging of their brain and bladder tissues, along with immunohistological staining, was also performed.
We observed significant increases in NLRP3/caspase-1-mediated inflammation following VEGF instillations, persisting over a week. This inflammation was present in both bladder and brain tissues. IC mice also showed increased pain sensitivity and voiding frequency. Additionally, inhibiting NLRP3/caspase-1 inflammasome activation with MCC950, a specific NLRP3 inhibitor, significantly reduced both inflammatory responses and pain and voiding behaviors. Furthermore, treatment with an orally active NLRP3 inhibitor, Usnoflast, showed improvements in reducing inflammation and abnormal behaviors. This research provides a translational foundation for understanding the role of inflammation in the development and progression of IC and suggests that targeting the NLRP3/caspase-1 signaling pathway could be a potential therapeutic approach.
The Role of NLRP3/Caspase-1-Related Inflammation in Interstitial Cystitis/Bladder Pain Syndrome (IC/BPS)
Hillarie Arellano , Michael Fletcher , Ashlyn Cochran , Kyle McClure , Alexander J. Li ,
Anna Malykhina , Fletcher White , Tyler Nguyen
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1,2,4
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¹School of Science, Indiana University Indianapolis, Indiana
²Department of Anesthesia, Indiana University School of Medicine, Indianapolis, Indiana
³Division of Urology, Department of Surgery, University of Colorado Denver, Anschutz, Aurora, Colorado
⁴Richard L. Roudebush VA Medical Center, Indianapolis, Indiana
Introduction
Interstitial cystitis/bladder pain syndrome (IC/BPS) is a chronic condition marked by chronic pelvic pain and urinary dysfunction, including increased urgency, frequency, and sleep-disturbing nocturia¹. Its underlying mechanisms are not fully understood, and diagnosis is still largely one of exclusion². These limitations highlight the need for mechanistically targeted interventions grounded in the biology of bladder inflammation and pain. Elevated serum levels of pro-inflammatory cytokines IL-1β and IL-18 in IC/BPS patients are consistent with NLRP3/caspase-1 inflammasome activation³⁻⁵. A clinically safe, orally active NLRP3/caspase-1 inhibitor, therefore represents a promising targeted therapeutic approach.
Accumulating evidence implicates innate immune signaling and neuroimmune crosstalk in the pathophysiology of IC/BPS. Danger- and pathogen-associated molecular patterns (DAMPs/PAMPs) can engage pattern recognition receptors and trigger NF-κB-dependent “priming” and subsequent activation of the NLRP3 inflammasome in urothelial, immune, and neural cells⁶⁻⁷. Upon activation, NLRP3 recruits the adaptor ASC and pro-caspase-1 to form a multiprotein complex that cleaves pro-IL-1β and pro-IL-18 into their mature, proinflammatory forms and initiates pyroptotic cell death, among other downstream events⁸⁻¹⁰. This signaling cascade may amplify local inflammation, compromise urothelial barrier integrity, and drive peripheral and central sensitization, providing a plausible mechanistic link between sterile bladder inflammation and chronic visceral pain.
Targeting NLRP3 therefore represents a
rational strategy to interrupt this maladaptive
inflammatory loop. Usnoflast (ZYIL1) is a novel,
orally available, small-molecule, selective
NLRP3 inflammasome inhibitor¹¹. It is highly
potent in human whole-blood assays and has
been shown to suppress NLRP3-driven
inflammation with demonstrable distribution
to brain and cerebrospinal fluid in nonclinical
species¹¹⁻¹². Usnoflast has achieved clinical
proof-of-concept in amyotrophic lateral
sclerosis and, as of November 2025, is
being evaluated in a phase 2a trial
(NCT05981040). However, its potential to
modulate bladder-driven neuroinflammation
and visceral pain has not been explored.
Here, we use an established VEGF-A-induced murine model of bladder pain syndrome in caspase-1 luciferase reporter mice to test whether systemic NLRP3 inhibition with usnoflast can attenuate inflammasome activation in the CNS and PNS and reduce pain-related outcomes. We combine in vivo bioluminescence imaging of caspase-1 activity with behavioral measures of bladder-associated pain and immunofluorescent labeling of inflammasome and glial markers in bladder and brain tissues. Together, this approach is designed to test the hypothesis that NLRP3-driven inflammasome signaling contributes to IC/BPS-like pathology and that pharmacologic NLRP3 inhibition with usnoflast can interrupt this pathway to alleviate bladder pain and associated inflammatory changes.
Figure 1: Priming and Assembly of the NLRP3 Inflammasome Pathway.
Caspase-1 Biosensor Model
Figure 2: PAMPs/DAMPs trigger NLRP3 inflammasome assembly, driving autocatalytic cleavage of pro-caspase-1 and concomitant activation of the transgenic luciferase reporter in biosensor mice.
Methods
To monitor inflammasome activity in vivo, we employed a transgenic caspase-1 luciferase biosensor mouse line, in which caspase-1 activation drives a luciferase-based bioluminescent signal. This design provides a quantitative, noninvasive readout of inflammasome activation, largely reflecting NLRP3-caspase-1 signaling under our experimental conditions.
Mice were housed under standard conditions with ad libitum access to
food and water and maintained on a 12 h light/dark cycle. All procedures
were approved by the Institutional Animal Care and Use Committee and
conformed to NIH guidelines for the care and use of laboratory animals.
A bladder pain state was induced by intravesical instillation of
VEGF-A. Transurethral instillations were performed three times
over the course of the experiment to establish a persistent bladder
inflammation and pain phenotype. Mice were randomly assigned
to experimental groups receiving vehicle (saline), VEGF-A alone, or
VEGF-A followed by the NLRP3 inhibitor usnoflast (10 mg/kg, intravesical administration).
Experimental Timeline
Figure 3: Experimental Timeline: Thinned-skull windows are prepared 2 weeks before baseline (day 0). VEGF-A bladder instillations are administered on days 1, 3, and 5. IVIS imaging, void spot assay (VSA), and pain-related behavioral testing are conducted 24 hours after each instillation, and at 3 days and 1 week after the third instillation. Bladder and brain tissues are collected following final assessments on day 12.
IVIS Imaging
To monitor inflammasome activation, we employed a caspase-1-dependent luciferase biosensor mouse line, in which caspase-1 activation drives a bioluminescent signal. Mice received an intraperitoneal injection of D-luciferin and were anesthetized with isoflurane for imaging. Whole-body bioluminescence was acquired using an IVIS system at defined time points before and after VEGF-A instillation and treatment. Regions of interest encompassing the lumbosacral spinal cord and pelvic region were drawn for each image, and average radiance (photons/s/cm²/sr) was quantified as a noninvasive, longitudinal readout of caspase-1-dependent inflammasome activity. Radiance values were normalized to baseline for within-subject comparisons across time and treatment.
Void Spot Assay (VSA)
To measure urinary frequency, mice were briefly housed on absorbent filter paper in clean cages for three consecutive 10-minute sessions at baseline and at the same time points as the pain tests. Void spots on filters were visualized under blacklight and imaged to measure voiding frequency and total voiding area. Voiding outcomes were analyzed separately by sex and treatment group to assess sex-specific and drug-related effects on micturition behavior.
Figure 4: Schematic illustration of in vivo assessments used in the VEGF-A–induced bladder pain model: (top left) IVIS bioluminescence imaging of caspase-1–dependent luciferase activity to quantify inflammasome activation; (bottom left) void spot assay (VSA) to evaluate urinary frequency and patterning; (top right) abdominal von Frey testing to assess referred mechanical hypersensitivity; and (bottom right) mouse grimace scale scoring to quantify spontaneous, ongoing pain.
Pain Behavior Testing
Mechanical hypersensitivity was assessed using calibrated von Frey filaments applied to the pelvic region. Mice were acclimated to testing chambers before measurements, and filaments were applied in ascending order of force using a standardized up-down response method to determine withdrawal thresholds. Mean scores were statistically compared across conditions to evaluate the impact of treatment on pain sensitivity.
Spontaneous pain-like behavior was quantified using the Mouse Grimace Scale (MGS)¹³. Mice were acclimated to the testing room and recorded individually in transparent chambers under ambient lighting. Still images of each animal’s face were extracted at predefined time points for offline scoring. Images were scored by an observer blinded to treatment using the five standard MGS action units: orbital tightening, nose bulge, cheek bulge, ear position, and whisker change. Each action unit was assigned a score of 0 (not present), 1 (moderately visible), or 2 (obviously present), and scores were averaged across action units to generate a composite MGS score for each mouse at each time point. Group means were then compared across conditions to evaluate treatment effects on ongoing, affective components of pain.
Tissue Collection
Mice were deeply anesthetized and perfused one week after the third VEGF-A instillation. Brains and bladders were dissected, fixed in 4% paraformaldehyde, cryoprotected in OCT Compound, and stored at -80°C for subsequent immunofluorescent and Western Blot analysis.
Statistical Analysis
Bioluminescence, von Frey, Mouse Grimace Scale, and void spot assay data were analyzed using parametric statistical tests appropriate for each comparison (e.g., Student’s t-tests or one- and two-way ANOVA), as specified in the figure captions. Statistical significance was defined as α = 0.05 unless otherwise indicated, and data are presented as mean ± SEM.
Results
Repeated intravesical exposure to VEGF-A induced robust caspase-1-dependent bioluminescence signals in brain and abdominal regions.
Repeated intravesical VEGF-A instillation induced robust caspase-1–dependent bioluminescence in both brain and abdominal regions. Compared with saline-treated controls, caspase-1–mediated signals were significantly elevated 24 h after the second and third instillations, indicating sustained inflammasome activation with repeated VEGF-A exposure.
Figure 5: Repeated VEGF-A instillations induce robust caspase-1 activation in the brain and abdominal regions (red circles) of female mice after the 2nd and 3rd instillations. (n=5/group; ****p<0.0001, Two-way ANOVA, Tukey’s HSD)
To confirm NLRP3 dependence of VEGF-A-driven inflammasome activity in our model, we first used the tool compound MCC950, a selective NLRP3 inhibitor. Female caspase-1 reporter mice were subjected to either 3x saline, 3x VEGF, or 3x VEGF + subsequent MCC950 (IP) treatment. Fresh bladder tissues and brain slices were collected 24 h after the third instillation, maintained in an oxygenated bath, and incubated with D-luciferin for ex vivo bioluminescence imaging
NLRP3 inhibition via systemic MCC950 reduces caspase-1 activation ex vivo.
Figure 5: NLRP3-inhibition with MCC950 reduced IC-related inflammation in female mice. (Left) Sample images shows both ex vivo bladder and brain tissue caspase-1 signals. (Right) VEGF instillation significantly increased both bladder and brain caspase-1 inflammatory IVIS signal, which were highly suppressed in MCC950 treated group. (n=5/group; *p<0.05, ***p<0.001. ****p<0.0001, One-way ANOVA, Tukey’s HSD).
Intravesical usnoflast demonstrates in vivo potential to reduce NLRP3 activity.
In a parallel pilot experiment in males (n=2 per group), in vivo bladder exposure to usnoflast produced a consistent reduction in VEGF-A–evoked caspase-1 signal compared with vehicle, although this effect did not reach statistical significance in this underpowered dataset. The clear downward trend in the usnoflast-treated bladders nevertheless supports the feasibility of pharmacologic NLRP3 inhibition in this model and motivates larger studies to rigorously define its impact on inflammasome activity.
Bladder
Brain
Figure 6: Usnoflast-treated male mice show a downward, but non-significant, trend in VEGF-A-evoked caspase-1 signal in vivo (pilot, n = 2 per group).
NLRP3 inhibition attenuated VEGF-induced pain responses
MCC950: NLRP3 Inhibition Attenuates Mechanical Hypersensitivity and Spontaneous Pain in Female Mice
Pharmacologic NLRP3 inhibition with MCC950 attenuated VEGF-induced pain behaviors in female mice. VEGF instillation produced robust mechanical hypersensitivity on abdominal von Frey testing, reflected by a significant decrease in withdrawal threshold 24 h after the first instillation and persisting for at least one week after the third instillation compared with saline controls. MCC950 treatment significantly increased withdrawal thresholds across these time points, indicating attenuation of VEGF-induced mechanical hypersensitivity.
Mouse grimace scores, assessed in the same female mice, were significantly elevated by VEGF and remained increased for at least 1 week after the final instillation; MCC950 significantly reduced grimace scores at 3 days and 1 week post-instillation.
Usnoflast: Effects on Spontaneous Pain in Male Mice
In males, VEGF similarly increased spontaneous pain, as evidenced by significantly elevated mouse grimace scores that were attenuated by usnoflast. In contrast, abdominal von Frey thresholds in the male cohort did not differ significantly between groups and are therefore not shown.
Figure 7: MCC950 significantly reduce grimace pain score at 3 days and 1 week after the last instillation. Both plots: n=5/group; *p<0.05 for Saline vs. VEGF, #p<0.05 for VEGF vs. VEGF+MCC950, Two-way ANOVA, Tukey’s HSD
Figure 8: Usnoflast transiently attenuated VEGF-induced spontaneous pain in male mice, with significantly reduced mouse grimace scores 24 h after the third instillation in the VEGF+Usnoflast group compared with VEGF alone (n = 5 per group; *p<0.05, Two-way ANOVA, Tukey's HSD); no other time points reached significance.
Usnoflast decreased IC/BPS-associated micturition frequency in females compared to males.
Voiding behavior assessed by void spot assay revealed a sex-dependent effect of usnoflast on IC/BPS-associated micturition frequency. In females, VEGF instillation increased the number of voids compared with saline controls, consistent with an IC/BPS-like urinary phenotype. Usnoflast treatment significantly reduced voiding frequency 24 h after the second and third instillations and again at 1 week after the third instillation, whereas effects at 3 days post-third instillation did not reach statistical significance. In contrast, in males, VEGF-induced changes in voiding frequency were not significantly altered by usnoflast at any time point, suggesting potential sex-related differences in IC/BPS-associated urinary pathology and treatment responsiveness.
Figure 9: Usnoflast instillations alleviated VEGF-induced voiding dysfunction in a small female cohort. (n=3-4/group; *p<0.05 for Saline vs. VEGF, #p<0.05 for VEGF vs. VEGF+ Usnoflast, Two-way ANOVA, Tukey’s HSD)
Discussion
VEGF-A-driven bladder inflammation in this model was associated with robust caspase-1-dependent bioluminescence in abdominal and brain regions, with signals significantly elevated 24 h after the second and third intravesical instillations. In female mice, these molecular changes coincided with marked abdominal mechanical hypersensitivity and increased Mouse Grimace Scale scores, indicating both evoked and spontaneous pain. Pharmacologic NLRP3 inhibition with MCC950 significantly attenuated VEGF-A-induced decreases in von Frey thresholds and reduced grimace scores at later time points, supporting a functionally important role for NLRP3-caspase-1 signaling in VEGF-A-evoked bladder pain.
Voiding behavior broadly paralleled the pain phenotype in females. VEGF-A increased micturition frequency on the void spot assay, consistent with an IC/BPS-like urinary phenotype, and usnoflast treatment reduced voiding frequency at several time points, partially normalizing VEGF-A-induced changes. In males, VEGF-A also increased spontaneous pain, and usnoflast produced a transient reduction in grimace scores 24 h after the third instillation; however, abdominal von Frey thresholds and voiding frequency did not differ significantly between male treatment groups. Pilot ex vivo experiments in male bladder tissue showed a consistent but non-significant downward trend in VEGF-A-evoked caspase-1-dependent signal with usnoflast. While the sample size limits interpretation, the convergence of in vivo grimace and ex vivo bioluminescence trends, together with the sex-dependent voiding data, suggests that usnoflast may modulate inflammasome activity and that IC/BPS-related pathology and/or treatment responsiveness in this model may differ by sex, justifying larger, adequately powered studies.
A major strength of this work is the use of a caspase-1–dependent bioluminescent reporter to functionally link inflammasome activation with pain-related behaviors across peripheral and central tissues. However, the reporter’s global nature precludes assigning signals to specific cell populations, and immunofluorescence and Western blot analyses of NLRP3 pathway markers from collected brain and bladder tissue are still in progress. Additional limitations include modest sample sizes, particularly in male and ex vivo cohorts, the lack of direct cytokine quantification (e.g., IL-1β, IL-18), and potential hormonal influences that were not explicitly controlled (e.g., estrous cycle stage in females). Despite these constraints, the data support NLRP3 as a plausible therapeutic target in IC/BPS and lay the groundwork for mechanistic follow-up studies and eventual translational testing of NLRP3-directed interventions, including usnoflast, in human IC/BPS tissue and early-phase clinical settings.
Future Directions
Immunofluorescence and Western Blot Assay
In the brain, immunofluorescence will be used to assess both glial activation and inflammasome-related signaling. Sections are stained with DAPI, GFAP, and IBA1 to visualize nuclei, astrocytes, and microglia, respectively, and in a separate series with DAPI, NLRP3, and caspase-1 to localize inflammasome components. In the bladder, sections were stained with DAPI, NeuN, and caspase-1 to examine neuronal populations and caspase-1 expression within the bladder wall. Analysis of these images is ongoing, and will compare regional and cell type-specific NLRP3/caspase-1 expression across saline-, VEGF-, and VEGF+NLRP3 inhibitor-treated groups, linking cellular sources of inflammasome activation to the bioluminescent and behavioral results above.
IVIS Imaging
Our current model uses a globally expressed caspase-1–dependent luciferase reporter line, which provides high-sensitivity readouts of inflammasome activity but limited cellular resolution. Future studies could incorporate next-generation biosensors, including cell type–restricted caspase-1 reporters and constructs that emit signal only in defined biochemical contexts (e.g., caspase-1 activation in the presence of other molecules). Coupling these tools with longitudinal in vivo and ex vivo IVIS imaging would allow more precise mapping of where, when, and in which cell populations inflammasome activation emerges in relation to bladder inflammation and pain behaviors.
Human IC Tissue Testing
Should these preliminary findings be confirmed in larger studies, the next step will be to test Usnoflast ex vivo on freshly biopsied bladder tissue from patients with IC/BPS, assessing its effects on inflammasome and inflammatory signaling by western blot and immunofluorescence (e.g., caspase-1, NLRP3, and downstream cytokines). These experiments would begin to establish the translational relevance of NLRP3 inhibition in human tissue. A key limitation is that biopsy samples would necessarily be obtained from patients with an established clinical diagnoses, and thus primarily represent chronic stage IC/BPS, which may not fully capture the drug's effects in earlier or initiating phases of the condition.
Conclusions
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VEGF-A instillation in this mouse model produced a reproducible IC/BPS-like phenotype, including caspase-1-dependent bioluminescence in abdominal and brain regions, mechanical hypersensitivity, increased grimace scores, and increased micturition frequency.
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Pharmacologic NLRP3 inhibition with MCC950 significantly reduces VEGF-induced caspase-1 activity and bladder pain behaviors, supporting a key role for NLRP3 inflammasome signaling in this model.
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Usnoflast (ZYIL1), a novel orally active NLRP3/caspase-1 inhibitor, demonstrates early anti-inflammatory and symptom-relieving effects, particularly in female mice, including reductions in neuroinflammation, micturition frequency, and pelvic pain.
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Observed differences between females and males in voiding outcomes suggest that IC/BPS-like pathology and/or responsiveness to NLRP3-targeted interventions may differ by sex in this model, underscoring the importance of sex as a biological variable in future mechanistic and translational studies.
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Collectively, these findings support NLRP3 inhibition as a plausible therapeutic target for IC/BPS and lay the groundwork for follow-up studies integrating cell-specific analyses, cytokine measurements, and eventual testing of NLRP3-directed compounds such as usnoflast in human IC/BPS tissue and early-phase clinical settings.
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