Centripetal structural reorganization of the testis in experimental rheumatoid arthritis and its age-dependent restitution by meloxicam: A morphometric and immunohistochemical study in rats.

BACKGROUND: Rheumatoid arthritis (RA) is a systemic autoimmune disease increasingly implicated in male reproductive impairment, yet the structural mechanism, spatial sequence and age-dependence of testicular injury-and whether it can be pharmacologically prevented-remain undefined. We characterized the morphological, histochemical, morphometric and immunohistochemical reorganization of the testis in experimental RA in two age groups and tested the protective effect of the preferential cyclooxygenase-2 inhibitor meloxicam.

MATERIALS AND METHODS: One hundred and twenty sexually mature male outbred white rats of two ages (8 and 18 months) were randomized, with stratification by age, to intact control, experimental RA (single subcutaneous 0.1 mL complete Freund's adjuvant at the tail base), or RA + meloxicam (1 mg/kg, oral gavage, once daily for 14 days). After simultaneous euthanasia on day 29, testes underwent organometry, haematoxylin-eosin and van Gieson picrofuchsin histology, and morphometry by the Autandilov method (≥30 transversely sectioned tubules per animal, sampled in the subcapsular, intermediate and central zones). Immunohistochemistry for Ki-67 (rabbit monoclonal, clone 30-9) and vimentin (mouse monoclonal, clone V9) was performed on a Ventana BenchMark XT automated stainer with ultraView Universal DAB detection and quantified in QuPath 0.5.1, with the animal as the biological replicate. With the animal as the experimental unit, data were analysed by two-way ANOVA (age × treatment) with Holm-corrected post-hoc tests, Student's t-test or Mann-Whitney U-test, and effect-size estimation (Hedges' g); p < 0.05 was considered significant.

RESULTS: In RA, the changes comprised microvasculitis and mucoid swelling of the tunica albuginea and interlobular septa and were associated with loss of Leydig cells and thinning of the spermatogenic epithelium. Tinctorial changes and tubular atrophy decreased from the subcapsular zone toward the centre, consistent with a centripetal (subcapsular-to-central) gradient. Tunica thickness increased by 27.5% (8 months) and 39.6% (18 months); the tubular outer diameter fell while the lumen widened, mathematically indicating epithelial thinning, and spermatocytes were the most affected population. Leydig-cell density fell by 10.8% and 32.6%. Vimentin (14.7→29.5% and 17.7→45.3%) and the Ki-67 index (18.3→33.8% and 15.1→30.7%) rose significantly (p < 0.05-0.001). Two-way ANOVA showed a significant treatment effect for all endpoints and a significant age × treatment interaction for outer diameter and Leydig-cell density; effect sizes were large (Hedges' g up to 2.8), and injury was 1.4-3.3-fold greater in aged rats. Meloxicam attenuated the vascular-stromal changes, repopulated Leydig cells and returned both markers toward baseline; restitution was partial and more evident in young than in aged animals.

CONCLUSION: In experimental RA the testis undergoes an ordered, centripetal reorganization in which germinal changes are accompanied by vascular-stromal alterations; age is an independent determinant of both injury severity and recovery, the ageing gonad entering the disease with a halved Sertoli-cell reserve. The preferential COX-2 inhibitor meloxicam was associated with morphological protection of the testis in this rat model, and a quantitative panel of tunica thickness, the outer-diameter-to-lumen ratio, Leydig-cell density and Ki-67/vimentin expression may help identify dysfunction at a potentially reversible stage.