Anti-inflammatory iron chelator, DIBI, reduces leukocyte-endothelial adhesion and clinical symptoms of LPS-induced interstitial cystitis in mice

Abstract

BACKGROUND: Interstitial cystitis (IC) is a prevalent and debilitating chronic inflammatory disease of the urinary bladder. Currently there are no fully effective therapeutic agents available, in part due to the still obscure pathogenesis of IC. Lipopolysaccharide (LPS) also known as endotoxin from Gram negative bacteria elicits IC in mice and has formed the basis of model systems for investigation. Excess free iron plays an important role in inflammation through generation of reactive oxygen species (ROS). The novel iron chelator DIBI has been shown to sequester excess free iron and dampen excess inflammatory responses to systemic LPS administration and also to Gram negative bacterial infections. OBJECTIVE: The overall objective of this study was to evaluate the effects of DIBI on LPS induced IC in mice. Leukocyte activation, endothelial adhesion and functional capillary density were assessed by intravital microscopy of the bladder microcirculation following a single intravesical LPS administration with or without intravesical DIBI treatment. Clinical IC symptoms were also assessed through behavioral and pain threshold force measurements. METHODS: Four groups of female BALB/c mice (n = 5–6/group) were randomized in this study: control group, IC group without therapy, IC group with DIBI therapy and control group with DIBI therapy. The groups were examined using intravital microscopy (IVM) of the bladder for leukocyte-endothelial interactions (adherent leukocytes, temporarily interacting leukocytes) and functional capillary density (FCD). A modified behavioral score by Boucher et al. and Von-Frey-Aesthesiometry were used to evaluate key behavioral indices related to pain and visceral pain perception. RESULTS: LPS introduced intravesically induced an early (≤2h) inflammation of the bladder evidenced by leukocyte activation and adhesion to bladder capillary walls. Intravesical DIBI therapy of mice 30min following LPS administration and assessed after 1.5h treatment showed a significant decrease in the number of adherent leukocytes compared to IC animals without DIBI treatment. DIBI treated mice showed a significantly lowered increase in behavioral distress scores compared to IC mice without therapy. Untreated IC mice exhibited a significantly decreased threshold force value for evoked pain response and DIBI treatment improved the threshold pain response. A significant inverse correlation was found for the two pain and suffering evaluation methods results. CONCLUSION: DIBI reduced inflammatory endothelial leukocyte adhesion and key indices related to pain and suffering over those observed in untreated IC mice. Our findings suggest a potential therapeutic role for DIBI for IC treatment.

Intravital Microscopy of Lipopolysaccharide-Induced Inflammatory Changes in Different Organ Systems—A Scoping Review

“…Berger et al found intravesical LPS administration to be superior at increasing adhesion when compared to intraperitoneal LPS administration [49]. The other studies examined exclusively used intravesical administration for LPS [6,47]. This is consistent with work comparing intravesical and intraperitoneal LPS administration in rats.…”

“…Interestingly, both groups used the same method for intravesical LPS administration: 150 µg/mL of LPS in 50 µL of saline held in the bladder for 30 min. They also both used FITC-albumin for visualization of the capillary beds, and both began imaging two hours after LPS administration [6,49]. [6]…”

“…The administration of LPS occurs prior to imaging, but the time between LPS administration and imaging varied slightly between groups. Kowalewska et al began imaging 4 h post-LPS administration [47], while both Hagn et al and Berger et al began their imaging only 2 h post-LPS administration [6,49]. Despite these differences, these timepoints are all considered acute models of inflammation.…”

“…Red blood cells lack mitochondria, allowing leukocytes to be visualized when fluorochromes are excited with light in the 515-560 nm range [5]. Using a combination of both fluorochromes allows for studies of leukocyte-endothelial interactions and microvascular perfusion following an inflammatory challenge [6][7][8].…”

Anti-Inflammatory Effects of the Iron Chelator, DIBI, in Experimental Acute Lung Injury

“…Although DIBI has been shown to have therapeutic effects in local and systemic inflammatory models [11][12][13][14][15], the molecular mechanisms involved in these effects have yet to be fully understood. Therefore, we established an experimental ALI model in mice by using intranasal endotoxin administration at a dosage of 5 mg/kg LPS and aimed to study the effects of DIBI at several treatment timepoints post LPS administration.…”