Interstitial cystitis/bladder pain syndrome (IC/BPS) is a chronic condition involving bladder inflammation, characterized by pelvic pain originating from the urinary bladder, along with increased urinary frequency and urgency. Hyaluronic acid (HA) bladder infusion has been proposed as a feasible treatment for IC/BPS by restoring the glycosaminoglycan layer on urothelium. Besides, HA is also recognized for its therapeutic potential in modulating inflammatory responses. High molecular weight HA (HMW-HA), typically defined as HA with a molecular weight exceeding 1000 kilodaltons (kDa), has demonstrated anti-inflammatory effects through CD44 binding, particularly in joint osteoarthritis and liver fibrosis [1,2]. In this study, we proposed a novel approach for IC/BPS treatment by directly injecting HMW-HA into the bladder wall of a rat model of interstitial cystitis (IC) induced by cyclophosphamide [3], hypothesizing that HMW-HA could reduce bladder inflammation, improve bladder functional outcomes, and alleviate central sensitization of pain signals (Figure 1).

Female Sprague-Dawley rats, 7 weeks old, were randomized into four study groups: sham (treated with HMW-HA), IC + saline, IC + normal molecular weight HA (N-HA), and IC + HMW-HA. The IC rat model was induced by intraperitoneal injection of cyclophosphamide at 75mg/kg on experimental days 0, 3, and 6. Rats in the sham control group received intraperitoneal injection of physiological saline on the same schedule. Pre-treatment mechanical hyperalgesia tests for hind paw were conducted on experimental day 7. Treatments with bladder wall injections (4 points, each point 50 μL) of either N-HA (molecular weight: 600-1200 kDa, dilute with normal saline to reach 1mg/ml), HMW-HA (molecular weight: > 1500 kDa, dilute with normal saline to reach to 1mg/ml), or physiological saline, were done on experimental day 7 depending on their assigned groups. Post-treatment functional evaluations, including bladder urodynamic assessments and post-treatment mechanical hyperalgesia tests for hind paw were done on experimental day 14 for functional outcome evaluation. All rats were euthanized after then, and their urinary bladders were harvested for Immunofluorescence analysis and western blotting to evaluate the quantitative data of pro-inflammatory cytokines interlukin-6 (IL-6) and tumor necrosis factor-α (TNF-α).

(1)	The levels of pro-inflammatory cytokines IL-6 and TNF-α were significantly elevated in the bladder tissue after IC induction. Bladder wall injections of HMW-HA markedly reduced the levels of these pro-inflammatory cytokines, whereas N-HA demonstrated only minimal effects. 
(2)	The urodynamic evaluation revealed significant bladder storage function improvements, including longer inter-contracting intervals and less episodes of non-voiding contractions, in the IC + HMW-HA group but not in the N-HA group. 
(3)	The pre-treatment withdrawal thresholds for hind paw were significantly reduced in all IC groups compared to the sham group. Post-treatment withdrawal thresholds revealed significant improvements after HMW-HA treatment, whereas N-HA treatment showed only marginal effect.

These findings suggested that HA can modulate bladder wall inflammation based on its molecular weight. Unlike traditional bladder infusion, direct injection of HMW-HA into the bladder wall effectively reduced pro-inflammatory cytokines IL-6 and TNF-α, whereas N-HA showed no significant effect. Additionally, bladder storage function improved significantly after HMW-HA injection, confirming its anti-inflammatory benefits.
Our IC model successfully demonstrated central sensitization of pain signals since the pre-treatment withdrawal thresholds over hind paw significantly reduced after IC induction. After treating with HMW-HA, the post-treatment withdrawal thresholds significantly improved, which indicated the anti-inflammatory effect on bladder wall alleviated the process of central sensitization.

Direct bladder wall injection of HMW-HA presents a promising treatment strategy for IC, offering both functional and molecular benefits that could improve patient outcomes.

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