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Abstract
Background. Concentrated acetic acid (vinegar essence, 70–80%) is the leading cause of corrosive ingestion across Russia, Eastern Europe and Central Asia, and produces not only local necrosis but a systemic phase of haemolysis, shock and multi-organ failure. Whether the pancreas — an organ outside the burn field - sustains injury at the level of apoptotic regulation has not been examined. Bcl-2 is the principal anti-apoptotic checkpoint of the mitochondrial cell-death pathway and is therefore the natural first marker to interrogate.
Objective. To quantify pancreatic Bcl-2 immunoexpression after a standardised 70% acetic acid burn of the digestive tract in rats of three ages, and to determine whether any change reflects true up-regulation in surviving cells or selective loss of Bcl-2-negative cells.
Methods. Ninety outbred albino rats aged 1, 3 and 6 months were assigned to intact control (n = 45) and chemical burn (n = 45), balanced across ages. Burn was produced by intra-oesophageal instillation of 0.5 mL of 70% acetic acid through a metal probe with 30–60 s exposure. Bcl-2 was demonstrated with DAB chromogen after citrate antigen retrieval; five fields per animal were scanned at ×200 and scored in QuPath 0.5.1. A total of 9149 nuclei were classified. The labelling index was compared by χ² with Yates’s correction, with odds ratios and Wilson 95% confidence intervals, and homogeneity of the effect across ages was tested by Woolf’s Q. Because a labelling index is a ratio whose denominator may itself change with injury, positive-cell, negative-cell and total nuclear counts were additionally normalised to the analysed field area.
Results. The Bcl-2 labelling index was higher after burn at every age: 26.13% → 36.56% at 1 month, 29.20% → 35.68% at 3 months and 26.67% → 39.44% at 6 months (all p < 0.001). Pooled, it rose from 27.47% (1395/5078) to 37.39% (1522/4071); χ² = 101.8, p < 0.001; odds ratio 1.58 (95% CI 1.44–1.72). The effect was not homogeneous across ages (Woolf Q = 7.61, df = 2, p = 0.022), so age-stratified reporting is required. Density analysis separated two distinct mechanisms. The density of Bcl-2-negative cells fell at every age (−36.5%, −30.6%, −16.7%), whereas the density of Bcl-2-positive cells was essentially unchanged at 1 and 3 months (+3.5%, −6.6%) and rose by 49.2% only at 6 months. The rise in labelling index at the two younger ages is therefore an arithmetic consequence of losing negative cells, while at 6 months it reflects genuine up-regulation. The pre-specified three-band grading scale classified every group, control and burned alike, as "moderate", and so detected none of this.
Conclusions. Caustic burn of the digestive tract measurably disturbs apoptotic regulation in the pancreas. Bcl-2-negative cells are selectively depleted at all ages, while a true anti-apoptotic response appears only in fully mature animals. A labelling index must not be reported without its denominator when the denominator is itself an outcome of the intervention — a caveat that applies well beyond this model.
Keywords:
Bcl-2, apoptosis, immunohistochemistry, digital pathology
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