Orchiectomy but not adjuvant-induced arthritis induces structural modifications in rat aortas

Abstract

Purpose: This study aimed to verify whether Adjuvant-Induced Arthritis (AIA) and/or Orchiectomy (ORX) modify the expression of the Nox1, Nox2 and Nox4 isoforms, the endothelial function or the structure of rat aortas.

Methods: Sixty-three Wistar rats were distributed into four groups: 1) Control; 2) ORX; 3) AIA; 4) Orchiectomy plus to Arthritis-induction (ORX/AIA). Thus, 21 days after the onset of AIA (by intradermal injection of Mycobacterium tuberculosis), the presence of Nox1, Nox2 and Nox4, the acetylcholine (ACh)-induced relaxation and the media layer thickness were assessed in the aorta taken from these animals.

Results: The Nox1, Nox2 and Nox4 were immunostained in intima, media and adventitia layers of aortas taken from all studied groups and AIA apparently increased this immunostaining. These modifications of Nox1, Nox2 or Nox4 expression, however, were not confirmed by Western blotting. In addition, neither AIA nor ORX changed the endothelial function, but ORX increased the media layer thickness in the studied aortas.

Conclusion: The present study showed weak clues of increased expression of Nox1, Nox2 and Nox4 as a result of AIA, as well as of Nox1 reduction caused by ORX. In addition, the endothelial function was not modified in the aortas of these animals by both AIA and/or ORX. On the other hand, ORX increased significantly the aorta media layer thickness in the studied animals, which was apparently mitigated by AIA.

Keywords: adjuvant-induced arthritis; aorta; histomorphometry; nicotinamide adenine dinucleotide phosphate (NADPH) oxidase; orchiectomy; rat.

Fig. 1.

Body mass (A) and wet mass of the left posterior paw (B), prostate (C) and seminal vesicle (D) obtained from animals from CTRL, ORX, AIA and ORX/AIA groups. Parametric data were compared by two-way ANOVA followed by the Tukey Post-Hoc test and values are expressed by mean ± S.E.M. (A). Non-parametric data were compared by the Mann-Whitney test, with critical values of P adjusted by Holm–Sidak post hoc correction and values expressed as median and interquartile range (25th first quartile; 75th third quartile; B–D). In parentheses is the number of samples. Superscript letters indicate statistically significant (P≤0.05) differences (a Compared to CTRL group; b Compared to ORX group; c Compared to AIA group). CTRL: control; ORX: orchiectomy; AIA: adjuvant-induced arthritis; ORX/AIA: orchiectomy in addition to adjuvant-induced arthritis.

Fig. 2.

Immunohistochemistry for Nox1, Nox2, and Nox 4 of thoracic aorta from CTRL, ORX, AIA and ORX/AIA groups. Nox isoforms-positive labels were observed in nuclei and/or cytoplasm of cells of the aorta layers, with immunoperoxidase staining in brown. Counterstaining with hematoxylin, which stains the background blue. Scale bar=50 µm. CTRL: control; ORX: orchiectomy; AIA: adjuvant-induced arthritis; ORX/AIA: orchiectomy in addition to adjuvant-induced arthritis.

Fig. 3.

Protein expression of the enzymes NADPH oxidases, isoforms Nox1 (A), Nox2 (B) and Nox4 (C) in relation to β-actin, determined in isolated thoracic aortas of animals from CTRL, ORX, AIA and ORX/AIA groups and densitometric quantification of the corresponding bands (Bars). Values expressed as median and interquartile range (25th first quartile; 75th third quartile). In parentheses is the number of samples. Comparisons between groups by Kruskal–Wallis’ test. CTRL: control; ORX: orchiectomy; AIA: adjuvant-induced arthritis; ORX/AIA: orchiectomy in addition to adjuvant-induced arthritis.

Fig. 4.

Concentration-response curves for acetylcholine (ACh) determined in isolated preparations of the thoracic aorta, obtained from animals belonging to the CTRL, ORX, AIA or ORX/AIA groups, in the absence (A) or presence of 10⁻⁴ mol/l apocynin (B). Relaxation was expressed as the percent of the pre-contraction induced by 10⁻⁵ mol/l norepinephrine. Points represent mean ± S.E.M. In parentheses, the number of samples. CTRL: control; ORX: orchiectomy; AIA: adjuvant-induced arthritis; ORX/AIA: orchiectomy in addition to adjuvant-induced arthritis.

Fig. 5.

Representative photomicrographs of thoracic aorta sections from CTRL, ORX, AIA and ORX/AIA groups, stained with hematoxylin and eosin, highlighting the media layer (A). al: adventitia layer; L: lumen. Symbols: arrow: endothelial cell. Scale bar=20 µm. Morphometry of the thickness of the media layer obtained in each experimental group (B). Data were compared by a two-way ANOVA test, followed by the Tukey Post-Hoc test, and values expressed by mean ± S.E.M. Superscript letters indicate statistically significant (P≤0.05) differences (a Compared to CTRL group; b Compared to AIA group; c Compared to ORX/AIA group). In parentheses is the number of samples. CTRL: control; ORX: orchiectomy; AIA: adjuvant-induced arthritis; ORX/AIA: orchiectomy in addition to adjuvant-induced arthritis.