Background & Aims
Patients diagnosed with Urologic Chronic Pelvic Pain Syndrome (UCPPS) suffer from poor quality of life due to the lack of adequate treatment for their pain (1). Research suggests that exposure to psychological stress increases the susceptibility to UCPPS, however, the exact pathophysiology of this relationship is unknown (2). We hypothesize that one potential biomarker that could explain the relationship between UCPPS and psychological stress is cell-free heme, a naturally occurring iron chelate located inside the protein hemoglobin (3,4). When a physical (or potentially psychological) insult to an animal or its tissues occurs, heme is liberated from red blood cells in a process known as hemolysis. Cell-free heme (heme that is not bound to proteins) acts as a pro-inflammatory agent which can result in tissue injury (5,6). In healthy individuals, extracellular heme is removed from plasma via hemopexin (7). However, little is known of the relationship between heme and UCPPS related pain.
Methods
Adult male and female Hpx knockout (KO) mice and wild type (WT) littermate controls were exposed to water-avoidance stress (WAS) for one hour to induce psychological stress. Control Hpx KO and WT mice were placed in the tank but no water was added. Pelvic and paw mechanical sensitivity was assessed using a simplified up-down method with von Frey monofilaments before and after exposure to WAS. Immediately after the exposure, blood plasma was collected and heme was quantified using spectrophotometry. The visceromotor responses (VMR) to balder distentions were measured in a separate cohort of female mice to assess bladder sensitivity. Additionally, bladder and paw samples were collected from mice to quantify Hpx, haptoglobin, and heme oxygenase-1 (HO-1, a heme scavenging enzyme) using western blot assay. All of the data analysis was done in Graph Pad PRISM (version 10.0.2).
Results
Analysis of behavioral data demonstrates a significant main effect of genotype with Hpx KO mice having more pelvic sensitivity (p<.001), but not paw sensitivity (p>.05), than their WT counterparts. No genotype by WAS interactions were observed for pelvic nor paw sensitivity (p>.05). However, a significant sex by WAS interaction demonstrates that exposure to psychological stress increases pelvic sensitivity (p<.05) in male Hpx KO and WT mice, but not female mice (p>.05) and not for paw sensitivity (p>.05). Female Hpx KO mice exposed to WAS showed increased VMR to bladder distensions which is indicative of increased pelvic sensitivity (p< .05). Although Hpx KO mice tended to have higher plasma cell-free heme concentration compared to WT, that difference was not statistically significant in either control or WAS conditions (p>.05). Protein quantification of Hpx, haptoglobin, and HO-1 is currently underway.
Conclusions
Hpx KO mice showed increased pelvic, but not paw, sensitivity suggesting that depletion of hemopexin has a tissue specific effect that contributes to increased nociception. Protein expression in discrete tissues will shed light on these differences and potentially explain why mice develop no sensitivity in one region but in another. We hypothesize that deep tissues might differ from cutaneous tissues in concentration of proteins like haptoglobin and HO-1, which are indirect markers of cell lysis. Furthermore, protein expression quantification might further explain why female Hpx KO mice exposed to WAS had an enhanced VMR but not increased mechanical sensitivity. Alternatively, increased pelvic sensitivity in Hpx KO mice could be heme-independent. As an anti-inflammatory mediator, depleted Hpx may promote inflammation. The quantification of inflammatory mediators will be conducted in future studies to better understand the relationship between Hpx and increased nociception.
References
1. Clemens, J. Q., Mullins, C., Ackerman, A. L., Bavendam, T., van Bokhoven, A., Ellingson, B. M., … & MAPP Research Network Study Group. Urologic chronic pelvic pain syndrome: insights from the MAPP Research Network. Nature Reviews Urology 2019; 16(3): 187-200.
2. Pierce, A. N., Di Silvestro, E. R., Eller, O. C., Wang, R., Ryals, J. M., & Christianson, J. A. (2016). Urinary bladder hypersensitivity and dysfunction in female mice following early life and adult stress. Brain Research 2016; 1639: 58-73.
3. Ferreira GC. Heme Synthesis. In: Lennarz WJ and Lane MD (eds) Encyclopedia of Biological Chemistry (Second Edition). Waltham: Academic Press, 2013, pp.539-542.
4. Xing Y, Gao S, Zhang X, et al. Dietary Heme-Containing Proteins: Structures, Applications, and Challenges. Foods 2022; 11 20221111.
5. Chiabrando D, Vinchi F, Fiorito V, et al. Heme in pathophysiology: a matter of scavenging, metabolism and trafficking across cell membranes. Frontiers in pharmacology 2014; 5: 61.
6. Pradhan P, Vijayan V, Gueler F, et al. Interplay of heme with macrophages in homeostasis and inflammation. International Journal of Molecular Sciences 2020; 21: 740.
7. Mauk MR, Smith A and Grant Mauk A. An alternative view of the proposed alternative activities of hemopexin. Protein Science 2011; 20: 791-805.
8. Adamian, L., Urits, I., Orhurhu, V., Hoyt, D., Driessen, R., Freeman, J. A., Kaye, A. D., Kaye, R. J., Garcia, A. J., Cornett, E. M., & Viswanath, O. A comprehensive review of the diagnosis, treatment, and management of urologic chronic pelvic pain syndrome. Current Pain and Headache Reports 2020; 24(6): 27.
9. Grinberg, K., Sela, Y., & Nissanholtz-Gannot, R. New insights about chronic pelvic pain syndrome (CPPS). International Journal of Environmental Research and Public Health 2020; 17(3005): 1-11.
10. Nickel, J. C., Teichman, J. M. H., Gregoire, M., Clark, J., & Downey, J. Prevalence, diagnosis, characterization, and treatment of prostatitis, interstitial cystitis, and epididymitis in outpatient urological practice: The Canadian PIE Study. Urology 2005; 66(5): 935-940.
11. Wingenfeld, K., Heim, C., Schmidt, I., Wagner, D., Meinlschmidt, G., & Hellhammer, D. H. HPA axis reactivity and lymphocyte glucocorticoid sensitivity in fibromyalgia syndrome and chronic pelvic pain. Psychosomatic medicine 2008; 70(1): 65.72.
12. Pierce, A. N., & Christianson, J. A. Chapter seventeen – Stress and chronic pelvic pain. Progress in molecular biology and translational science 2015; 131: 509-535.
Presenting Author
Anastasiia E. Gryshyna
Poster Authors
Anastasiia Gryshyna
MSc
University of Alabama at Birmingham
Lead Author
Felipe Vendrame
Ph.D.
University of Alabama at Birmingham
Lead Author
Karina Ricart
Ph.D.
University of Alabama at Birmingham
Lead Author
Ty Walker
B.S.
University of Alabama at Birmingham
Lead Author
Rakesh P. Patel
Ph.D.
University of Alabama at Birmingham
Lead Author
Jennifer DeBerry
Univ of Alabama at Birmingham
Lead Author
Topics
- Mechanisms: Biological-Systems (Physiology/Anatomy)