[Experimental optimization of paraffin sectioning techniques for the eyeball].

Abstract

Objective: To explore optimized protocols for paraffin section preparation of the eyeball to enhance the histological visualization of key ocular structures. Methods: It was an experimental research, conducted from September 2022 to September 2024. The first experiment involved 18 porcine eyeballs, which were divided into five groups (six subgroups) by the random number table method. Three fixatives were tested: Davidson's solution (Davidson group), a mixture of 1% paraformaldehyde and 1.25% glutaraldehyde (mixed solution group), and 4% paraformaldehyde (formaldehyde group). Each fixative was applied at room temperature and 4 ℃, respectively. Hematoxylin and eosin-stained paraffin sections were prepared and evaluated for basic quality and morphological conditions of the cornea, lens, anterior chamber angle, ciliary body, Zinn's zonule, sclera, and retina. In the second experiment, 33 porcine eyeballs were divided into 4 groups (11 subgroups) by the random number table method to assess the effects of different pre-fixation treatments. The section quality was scored and compared among the groups of commonly used clinical dissection methods, standard anatomical procedures, hemisected eyeballs, and untreated controls. The optimal pre-treatment and fixation conditions were then validated using six human eyeballs (three from elderly males and three from male adolescents). Statistical analyses were performed using the Kruskal-Wallis H test, Dunn test, Bonferroni correction, paired-sample t-test, and Mann-Whitney U test. Results: The Davidson group exhibited a rough globe surface, opacified lens, and blackened sclera. In contrast, the mixed solution group maintained a smooth surface, yellowing of the cornea and sclera, and lens transparency. The formaldehyde group also had a smooth surface, but the lenses appeared slightly opaque. The mixed solution group showed significantly higher scores for lens morphology (8.00±1.95) and Zinn's zonule (3.17±1.27) than the Davidson group (6.17±1.03 and 1.83±0.94, both P<0.05), and for Zinn's zonule and retinal morphology (5.58±1.83) compared to the formaldehyde group (1.50±0.67 and 2.92±0.79, both P<0.05). The anterior segment total score (27.67±4.74) was significantly higher in the mixed solution group than the Davidson group (22.83±2.98, P<0.05). The anterior segment total score and overall section quality score (40.58±7.53) were significantly higher in the mixed solution group than the formaldehyde group (23.17±3.04 and 32.42±4.01, both P<0.05). The 4 ℃ fixation condition yielded better preservation of Zinn's zonule (2.50±1.04) compared to room temperature fixation (1.83±1.29, P<0.05). Among the pre-treatment methods, the clinical approach group achieved higher scores in ciliary body (7.04±1.30), Zinn's zonule (2.96±0.95), sclera (6.63±1.38), retina (5.17±1.58), and total section quality (36.75±4.63), compared to hemisected specimens (all P<0.05). Application of the optimal pre-treatment method (0.5-cm corneal limbal incision) and fixation condition (mixed solution at 4 ℃) to human eyeballs successfully preserved and ensured clear visualization of the key structures, including the ciliary body, lens, Zinn's zonule, and ocular wall. Conclusions: The pre-treatment using a clinical method (e.g., corneal limbal incision), combined with 4 ℃ fixation in a paraformaldehyde-glutaraldehyde mixture, significantly enhances the preservation and visualization of essential ocular structures in paraffin sections. This method shows promise for improving the histopathological evaluation of the human eyeball.