Interstitial Cystitis/Bladder Pain Syndrome (IC/BPS) is a chronic condition with bothersome symptoms such as a compelling need to urinate due to pain or an unpleasant sensation that is difficult to defer [1]. It is hypothesized that damage to the glycosaminoglycan (GAG) layer contributes to (IC/BPS) symptoms, leading to the widespread use of intravesical GAG replenishment therapy for non-responsive patients. Despite numerous treatment options, there remains a necessity for definitive alternative therapies [2]. Building on this premise, our study aimed to investigate the histopathological and immunological effects of intravesical applications of Tarantula Cubensis Extract (TCE) and Alpha-linolenic Acid (ALA), known for their anti-inflammatory properties [3], in a chemical cystitis rat model with a comparison to the standard intravesical treatment, Hyaluronic Acid (HA).

Forty Wistar Albino female rats were utilized in the research. They were divided into five groups, each containing 8 rats: control, chemical cystitis (CS), CS+ALA, CS +TCE, and CS+HA. The experimental CS model was achieved by intraperitoneal instillation of cyclophosphamide (50 mg/kg, 0.7 mL) for a total of 3 doses every 3 days. The control group received 0.9% NaCl instillation. Among the treatment groups, 2 mL of 0.9% NaCl solution containing 200 mg/kg ALA was instilled in the CS+ALA group, 0.3 mL Theranekron (tarantula cubensis extract, 1:100 / D2, Richter Pharma, Austria) was instilled in the CS +TCE group, and 2 ml of HA (%0,5) was instilled in the CS+HA group. These agents were administered to the treatment groups twice weekly for two weeks. To create similar stress in the KS and control groups, 0.9% NaCl was instilled for the same period. The rats were euthanized by cervical dislocation under general anesthesia three days after the end of the treatments. Cystectomy was performed via abdominal incision. 
The bladder tissue was divided into two equal pieces. One-half of the bladder tissue was examined for histopathological changes such as mast cells, fibrosis, and degree of inflammation. Epithelial degeneration, fibrosis, submucosal edema, hyperemia, and inflammation were assessed using a scoring system from 0 to 3 (0 indicating no lesion, 1 for mild severity, 2 for moderate severity, and 3 for severe severity). Additionally, mast cells were counted across the entire section at a magnification of 20x. The other half was used for ELISA analysis to measure Interleukin 6 (IL6), Superoxide Dismutase 1 (SOD1), Malondialdehyde (MDA), Tumor Necrosis Factor Alpha (TNFα), and Nerve Growth Factor βeta (NGF-β) levels. IL-6, SOD1, MDA, TNF-α, and NGF-β molecules were examined using supernatants, respectively. After the test applications, the measurements were evaluated on a microplate reader.
The statistical analyses of the study were conducted using the IBM Statistical Package of Social Science version 29 (Chicago, IL, USA). Data were presented as mean ± standard error. One-way Analysis of Variance (ANOVA) was utilized to compare ELISA results among the study groups, while the LSD test was employed for multiple comparisons.

Three rats died during the study due to side effects and were excluded, resulting in a final sample size of 37 subjects. 
A significant increase in the degree of epithelial degeneration, submucosal edema, and inflammation was detected in the CS group compared to the control group. There was also a significant increase in the number of mast cells in the CS group compared to the control group. While a significant decrease in epithelial degeneration, fibrosis, and submucosal edema findings was observed in the CS+TCE and CS+HA groups, no significant difference was observed in the hyperemia and inflammation scores. A significant decrease was detected in the number of mast cells in both groups when compared to the CS group. In the CS+ALA group, a statistically significant improvement was detected in all findings except hyperemia (Table 1).
Immunologically, tissue MDA levels showed statistically significant differences according to the study groups. As a result of pairwise comparisons, it was determined that the lowest tissue MDA level was in the CS+ALA group, and this value was significantly lower than that of the CS+TCE and CS groups (p = 0.006). No statistically significant results were obtained for other parameters (Table 2).

The model's validity was confirmed by the increased degree of epithelial degeneration, submucosal edema, and inflammation and number of mast cells in the CS group than in the control group.
CS+TCE and CS+HA showed anti-inflammatory effects, with CS+ALA exhibiting the most significant improvements in multiple parameters, highlighting its potential as an intravesical treatment alternative.

ALA shows promise as an intravesical treatment option for IC/BPS, demonstrating anti-inflammatory effects and tissue preservation in our preclinical study. Further clinical investigations are warranted to validate its efficacy.

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