Molecular imaging of nuclear factor-κB in bladder as a primary regulator of inflammatory response

Abstract

Purpose: Nuclear factor-κB activation is implicated in chronic inflammatory disorders and it is a key regulator of genes involved in the response to infection, inflammation and stress. Interstitial cystitis and painful bladder syndrome are common inflammatory disorders of the bladder characterized by frequent urination and bladder pain. The role of nuclear factor-κB activation in bladder inflammation is not well defined.

Materials and methods: Female transgenic nuclear factor-κB-luciferase Tag mice (The Jackson Laboratory, Bar Harbor, Maine) were used to perform serial, noninvasive in vivo and ex vivo molecular imaging of nuclear factor-κB activation in the whole body after administering arsenic trioxide (5 mg/kg), lipopolysaccharide (2 mg/kg) or cyclophosphamide (Sigma®) (200 mg/kg) to initiate acute transient bladder inflammation. Pretreatment with dexamethasone (Sigma) (10 mg/kg) was used to modulate cyclophosphamide induced nuclear factor-κB dependent luminescence in vivo.

Results: Treatment of nuclear factor-κB-luciferase Tag mice with chemicals increased luminescence in a time and organ specific manner in vivo and ex vivo. The highest levels of bladder nuclear factor-κB dependent luminescence were observed 4 hours after cyclophosphamide administration. Pretreatment with dexamethasone 1 hour before cyclophosphamide injection significantly down-regulated cyclophosphamide induced bladder nuclear factor-κB dependent luminescence, ameliorated the grossly evident pathological features of acute inflammation and decreased cellular immunostaining for nuclear factor-κB in the bladder.

Conclusions: Nuclear factor-κB activity may have an important role in the pathophysiology of bladder inflammation. Nuclear factor-κB-luciferase mice can serve as a useful model in which to screen potential candidate drugs for cystitis associated with aberrant nuclear factor-κB activity. Such screening may significantly aid the development of therapeutic strategies to manage inflammatory bladder disorders.

FIGURE 1

Whole body in vivo imaging of NF-κB-Luc mice. (A) Animals received i.v. injection in the tail vein with lipopolysaccharide (LPS, 2 mg/kg), and i.p. injection with arsenic trioxide (As₂O₃, 5 mg/kg), and cyclophosphamide (CYP, 200 mg/kg); equivalent volume of phosphate-buffered saline, to serve as control. Images are presented after color scale adjustment. Gray scale images were obtained before luminescence imaging for reference. (B) Statistical analysis of total photon flux presented in time-fashion manner. White bars – controls (PBS); grey bars – As₂O₃; striped bars – LPS; black bars – CYP. The SD is indicated. * - p<0.05; ** - p<0.01 compared with controls. Details are described in ‘materials and methods’ section.

FIGURE 2

Ex-vivo NF-κB-dependent luminescence activity assessment of mouse kidney (A), skin (B), lung (C), and gut (D) after chemical administration. The images were taken 4 h after chemical administration and presented after color scale adjustment. Data represent the mean value of total photon flux from tissue studied after chemical administration. White bars – controls (PBS); grey bars – As₂O₃; strip bars – LPS; black bars – CYP. The SD is indicated.* - p<0.05; ** - p<0.01 compared with controls. Details are described in ‘materials and methods’ section.

FIGURE 3

Ex-vivo NF-κB-dependent luminescence activity assessment of mouse urinary bladder after chemical administration. The bladder images were taken 4 h after chemical administration and presented after color scale adjustment. (B) Statistical analysis of total photon flux is presented in time-fashion manner. Data represent the mean value of total photon flux from bladder after chemical administration. White bars – controls (PBS); grey bars – As₂O₃; strip bars – LPS; black bars – CYP. The SD is indicated. * - p<0.05; ** - p<0.01 compared with controls. Details are described in ‘materials and methods’ section.

FIGURE 4

Effect of dexamethasone on ex-vivo NF-κB-dependent luminescence activity of mouse urinary bladder after cyclophosphamide (CYP) administration. Mice were i.p. injected with dexomethasone (DEXO, 10 mg/kg), and CYP (200 mg/kg) was administered 1 h later to these animals and imaged after 4 h of treatment. (A) Whole body in vivo imaging, and (B) ex vivo bladder luminescence assessment. (C) Statistical analysis represents the mean value of total photon flux from the bladders. (D) Pre-treatment with dexamethasone ameliorates hyperemia which is a gross anatomic symptom of CYP-induced hemorrhagic cystitis. SD as indicated. White bars – controls (PBS); black bar – CYP, black and white bar – DEXO+CYP. * - p<0.05 compared with controls and DEXO+CYP. Details are described in ‘materials and methods’ section.

FIGURE 5

Assessment of NF-κB activity in the urinary bladder tissue 4 h after i.p. administration of a single dose (200 mg/kg) of cyclophosphamide (CYP). One hour before CYP administration some animals were i.p. injected with dexamethasone (DEXO) (10 mg/kg). (A) H&E histology and immunostaining of the bladder for NF-κB/p65 in representative samples from NF-κB/Luc transgenic mice. CYP induced significant tissue damage as evident by severe edema (black asterisks), leukocyte infiltration, and extensive epithelial loss (open arrowheads). Pre-treatment with DEXO reduced epithelial loss (black arrowheads) and edemas (black asterisk) in CYP injected animals. The strongest nuclear expression of NF-κB (dark brown color) was observed in microvessel endothelium (green arrows), extravasating leukocytes (red arrows), stromal scattered leucocytes (black arrows), and in the stromal accumulated leukocytes (inside black circle) after CYP administration. The staining was significantly diminished by dexamethasone (DEXO). Red dotted circles indicate microvessels. Left column presents bladder H&E staining. Right column depicts staining for NF-κB located inside of the black squares of the right column. (B) Box-plot analysis of absolute number of bladder cells positively stained only for NF-κB in the nuclei in the formalin-fixed paraffin-embedded whole mount mouse bladder specimens from animal treated with CYP. Results are expressed as absolute numbers of nuclear NF-κB positive cells in whole mouse bladder. The average is shown as a horizontal line through the box. The lower and upper margins of the box represent the 25^th and 75^th percentiles, with the extended arms representing the minimal and maximal number, respectively. The data were analyzed using one-way analysis of variance (ANOVA) with 95% confidence limits and Tukey’s correction. Details are described in ‘materials and methods’ section.

FIGURE 6