Mucosal sulfhydryl compounds evaluation by in vivo electron spin resonance spectroscopy in mice with experimental colitis

Abstract

Background: Sulfhydryl (SH) compounds are essential in maintaining mucosal integrity in the gastrointestinal tract. A decrease in colonic mucosal SH compounds affects the redox status of the mucosa, resulting in vulnerability to further attacks. Therefore, there is a strong need for in vivo evaluation of SH compounds in the colonic mucosa.

Aims: The aim of the current study was to establish a method of evaluating levels of SH compounds in the colonic mucosa of live animals before and after induction of colitis.

Methods: Murine experimental colitis was induced by instillation of trinitrobenzene sulphonic acid (TNBS) dissolved in 50% ethanol into the colon via the anus. For evaluation of mucosal SH compounds in the colon, 3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (carbamoyl-PROXYL), a stable nitroxide radical, was instilled into the colonic lumen of live mice and the spin clearance rate was measured by L-band electron spin resonance (ESR) spectroscopy.

Results: Morphological study showed that mucosal damage was severe one or two days after TNBS instillation. The colonic mucosa started to regenerate at four days, and looked normal at seven days, after induction of colitis. The spin clearance rate of carbamoyl-PROXYL decreased significantly at 0.5, 1, 2, and 4 days after induction of colitis compared with mice before TNBS instillation. Surprisingly, although the colonic mucosa looked normal seven days after TNBS administration, the spin clearance rate still remained significantly slow. The spin clearance rate returned to normal 14 days after induction of colitis. The change in in vivo spin clearance rate was consistent with the time dependent change in mucosal reduced glutathione, a major component of SH compounds.

Conclusion: The spin clearance rate obtained by L-band ESR spectroscopy in combination with carbamoyl-PROXYL can give an estimate of the level of colonic mucosal SH compounds in live animals and is useful for evaluating the mucosal defence system against oxidative stress.

Figure 1

(A) Photograph of the L-band electron spin resonance spectrometer system. (B) Schema of positioning the mice and the electromagnet. C57/BL6 male mice under pentobarbital anaesthesia were placed in the holder in an abdominal position. The proximal part of the hind leg of the mouse was placed at the axial centre of the loop gap resonator. The dotted lines represent the magnetic field gradient.

Figure 2

(A) Chemical structure of 3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (carbamoyl-PROXYL) and its corresponding hydroxylamine. The electron spin resonance (ESR) spectrum of carbamoyl-PROXYL has three hyperfine structure lines due to the N-14 nucleus. When carbamoyl-PROXYL is reduced to the corresponding hydroxylamine, this results in the disappearance of the ESR signal. (B) Decrease in ESR signal intensity after intracolonic instillation into a live mouse. The signal decay curve of carbamoyl-PROXYL was obtained by semi logarithmically plotting the peak heights of the ESR signal. The initial spin clearance rate was calculated from the slope of the signal decay.

Figure 3

In vivo electron spin resonance (ESR) measurement of 3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (carbamoyl-PROXYL) decay by L-band ESR spectroscopy. The peak of the low field component of the triplet signal is demonstrated. (A) The carbamoyl-PROXYL signal peaked initially and decayed rapidly. The ESR signal of carbamoyl-PROXYL cleared eight minutes after instillation. Subsequent instillation of ferricyanide (3 mM, 0.05 ml/mouse) into the colon caused the ESR signal to reappear. (B) N-ethylmaleimide (NEM) solution (2 mM, 0.1 ml/mouse) was instilled into the colonic lumen. Pretreatment with NEM inhibited ESR signal decay in an in vivo experiment.

Figure 4

Time course of changes in the colonic mucosa after trinitrobenzene sulphonic acid (TNBS) administration. (A–E) Representative photographs of the colons from mice given 1 mg of TNBS in 0.1 ml of 50% ethanol and killed at various times afterwards. (A) Normal colonic mucosa. (B, C) Colonic mucosa from a mouse one day (B) and two days (C) after TNBS administration. Acute colonic damage characterised by haemorrhage, ulcers, and bowel wall thickness was present. The ulcers were surrounded by thickened and inflamed mucosa. These colons were given a damage score of 5. (D) Colonic mucosa from a mouse four days after TNBS administration. Mucosal damage was still present, whereas the healing process had started in the colonic mucosa. (E) Colonic mucosa from a mouse seven days after TNBS administration. The colonic mucosa appears normal.

Figure 5

(A) Time course study of the spin clearance rate of 3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (carbamoyl-PROXYL) after trinitrobenzene sulphonic acid (TNBS) instillation into live mice. To help with the understanding of the relationship between mucosal damage and spin clearance rate, the graph of the time course observations of mucosal damage is given. Results are mean (SD) of five mice. There was a significant difference compared with mice without TNBS treatment: †††p<0.001, **p<0.005. (B) Time course study of mucosal reduced glutathione (GSH) concentrations after TNBS instillation into living mice. There was a significant difference from mice without TNBS treatment: †††p<0.001.