Novel application of low pH-dependent fluorescent dyes to examine colitis

Abstract

Background: Endoscopy capable of fluorescence observation provides histological information on gastrointestinal lesions. We explored the novel application of low pH-dependent fluorescent dyes for fluorescence observation of crypt structure and inflammatory cell infiltration in the colon.

Methods: Low pH-dependent fluorescent dyes were applied to the colonic mucosa of normal mice for observation under fluorescence stereomicroscopy system. We also examined mouse models of colitis, which were induced by trinitrobenzenesulfonic acid, dextran sulfate sodium or interleukin-10 deficiency.

Results: Topical application of low pH-dependent fluorescent dyes revealed crypts as ring-shaped fluorescent stains by visualizing the mucin granules of goblet cells. Because of the minimal fluorescence intensity of the low pH-dependent fluorescent dyes in phosphate-buffered saline, it was not necessary to wash the mucosa before the fluorescence observation. 4-Nitro-7-piperazino-2,1,3-benzoxadiazole (NBD-PZ) was quicker to achieve complete staining (three minutes) than LysoSensor Green DND-153 and DND-189 (20 minutes). In each type of colitis, NBD-PZ revealed the destruction of the crypts as the disappearance of the ring-shaped fluorescent stains and the infiltration of inflammatory cells as the aggregation of punctate fluorescent stains through visualization of lysosomes.

Conclusions: Low pH-dependent fluorescent dyes, especially NBD-PZ, are suitable for topical application to the colonic mucosa and have characteristics that allow for the histological examination of colitis.

Figure 1

Application of low pH-dependent fluorescent dyes to the mucosa of the normal colon. (A) HE stain and Alcian blue (pH 2.5) stain of the colonic mucosa obtained from a normal BALB/c mouse. Bar, 200 μm. (B) Stereomicroscopic observation of the colonic mucosa obtained from the normal BALB/c mouse. Bar, 200 μm. (C) Fluorescence stereomicroscopic observation of the colonic mucosa in PBS only. (D) Fluorescence stereomicroscopic observation of the colonic mucosa in PBS containing 1 μg/ml NBD-PZ after incubation at 37°C for three minutes. A magnified image of the indicated area is shown in the right panel. We observed five samples and obtained similar findings. (E) Stereomicroscopic and (F) fluorescence stereomicroscopic observation of the colonic mucosa in PBS containing 1 μg/ml LysoSensor Green DND-189 after incubation at 37°C for 20 minutes.

Figure 2

Application of NBD-PZ to the mucosa of TNBS-induced colitis. (A) Stereomicroscopic observation of the mucosa of TNBS-induced colitis. Bar, 200 μm. (B and C) Fluorescence stereomicroscopic observation of the mucosa of TNBS-induced colitis in PBS only (B) and in PBS containing 1 μg/ml NBD-PZ after incubation at 37°C for three minutes (C). (D) A magnified image of the indicated area in (C). We observed five samples and obtained similar findings. (E) HE stain of the colonic mucosa corresponding to (D). Bar, 200 μm. (F) Immunohistochemical stain of lysosomes using a section adjacent to the section shown in (E).

Figure 3

The effect of bafilomycin A1 treatment. The mucosa of TNBS-induced colitis was examined with 1 μg/ml NBD-PZ before and after 25 nM bafilomycin A1 treatment at 37°C for one hour.

Figure 4

Application of NBD-PZ to the mucosa of colitis induced by DSS or IL-10 deficiency. (A) Stereomicroscopic and (B) fluorescence stereomicroscopic observation of the mucosa of DSS-induced colitis in PBS containing 1 μg/ml NBD-PZ after incubation at 37°C for three minutes. A magnified image of the indicated area is shown in the lower panel. Bar, 200 μm. We observed five samples and obtained similar findings. (C) HE stain of the colonic mucosa corresponding to the indicated area in (B). (D) Stereomicroscopic and (E) fluorescence stereomicroscopic observation with 1 μg/ml NBD-PZ of the mucosa of IL-10 deficiency-induced colitis. A magnified image of the indicated area is shown in the lower panel. (F) HE stain of the colonic mucosa corresponding to the indicated area in (E).

Figure 5

Characterization of NBD-PZ-stained mononuclear cells isolated from the mucosa of colitis. (A) Mononuclear cells were isolated from the mucosa of TNBS-induced colitis and incubated with 0.25 μg/ml NBD-PZ at 37°C for three minutes for flow cytometry. A total of 10,000 cells were analyzed. The cells in the R1 region were defined as NBD-PZ-positive. (B) Mononuclear cells were isolated from the mucosa of TNBS-induced colitis, incubated with 0.25 μg/ml NBD-PZ at 37°C for three minutes, and reacted with anti-CD11b, CD3e or B220 antibody on ice for 30 minutes. A total of 10,000 NBD-PZ-stained cells were analyzed by flow cytometry and the cells in the M1, M2 or M3 region were defined as CD11b-, CD3e- or B220-positive, respectively. (C) Mononuclear cells were isolated from the mucosa of colitis induced by TNBS, DSS or IL-10 deficiency to characterize the NBD-PZ-stained cells under flow cytometry. Data represent the percentages of CD11b-, CD3e- and B220-positive cells within the NBD-PZ-stained cell pool, respectively (means ± standard deviations, n = 3).

Figure 6

Histological and biochemical examination of various organs following NBD-PZ enema. (A) HE stain of the colon obtained from a BALB/c mouse at 24 hours after intrarectal administration of 100 μl PBS containing 2.5 μg/ml NBD-PZ. Bar, 200 μm. (B) ALT and BUN concentrations in the plasma obtained from BALB/c mice at 24 hours after the enema of 2.5 μg/ml NBD-PZ. Data represent the means ± standard deviations (n = 5).

Figure 7

Mutagenicity test of NBD-PZ. (A and B) The umu-test bacterial strain, NM2009, was mixed with NBD-PZ (final 2.5 or 10 μg/ml), furylfuramide (0.03 μg/ml) or 2-aminoanthracene (0.3 μg/ml) in the absence (A) or presence (B) of liver homogenate, incubated at 37°C for two hours, and reacted with a chromogen. A compound is classified as mutagenic when it induces ≥ two times increase in absorbance at 670 nm compared with the sample that contains NM2009 only (the third or the first sample in A or B, respectively). Data represent the means ± standard deviations (n = 3).