Bcl-2 Immunoexpression In the Pancreas After Experimental 70% Acetic Acid Chemical Burn of The Digestive Tract: An Age-Stratified Quantitative Digital Immunohistochemical Study in Outbred Albino Rats

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

References

Chirica M, Bonavina L, Kelly MD, Sarfati E, Cattan P. Caustic ingestion. Lancet. 2017;389(10083):2041–2052. doi:10.1016/S0140-6736(16)30313-0

Hoffman RS, Burns MM, Gosselin S. Ingestion of caustic substances. N Engl J Med. 2020;382(18):1739–1748. doi:10.1056/NEJMra1810769

Ratcliffe A, Baker A, Smith D. Successful management of 70% acetic acid ingestion on the intensive care unit: a case report. J Intensive Care Soc. 2018;19(1):56–60. doi:10.1177/1751143717711062

Chibishev A, Simonovska N, Bozinovska C, et al. Severe renal function impairment in adult patients acutely poisoned with concentrated acetic acid. Arh Hig Rada Toksikol. 2012;63(1):47–54. PMID:23585201

Brusin K, Krayeva Y, Bushuev A, et al. Highly concentrated acetic acid poisoning: 400 cases reviewed. Asia Pac J Med Toxicol. 2012;1(1):3–8.

Acute pancreatitis after ingestion of acetic acid. Pancreas. 1996;12(3):320–321. PMID:8720672

Acute gastric necrosis induced by caustic substance ingestion: a case report. 2024. PMC11682535

Kaewlai R, Noppakunsomboon N, Tongsai S, et al. Performance of computed tomography and its reliability for the diagnosis of transmural gastrointestinal necrosis in a setting of acute ingestion of predominantly strong acid substances in adults. Clin Toxicol (Phila). 2023;61(5):346–354.

Anilkumar U, Prehn JHM. Anti-apoptotic BCL-2 family proteins in acute neural injury. Front Cell Neurosci. 2014;8:281. doi:10.3389/fncel.2014.00281

Bcl-2 expression in pancreas development and pancreatic cancer progression. J Pathol. 2001;194(4):444–450. PMID:11523052

Sung KF, Odinokova IV, Mareninova OA, et al. Prosurvival Bcl-2 proteins stabilize pancreatic mitochondria and protect against necrosis in experimental pancreatitis. Exp Cell Res. 2009;315(11):1975–1989.

Mitochondrial dysfunction and apoptosis of acinar cells in chronic pancreatitis. J Gastroenterol. 2008;43(10):795–803.

Angiotensin II mediates acinar cell apoptosis during the development of rat pancreatic fibrosis by AT1R. Pancreas. 2004;29(4):264–270. PMID:15502641

Latif N, Khan MA, Birks E, et al. Upregulation of the Bcl-2 family of proteins in end stage heart failure. J Am Coll Cardiol. 2000;35(7):1769–1777. doi:10.1016/S0735-1097(00)00647-1

Gordon R, Neal ML, Luo J, et al. Prokineticin-2 upregulation during neuronal injury mediates a compensatory protective response against dopaminergic neuronal degeneration. Nat Commun. 2016;7:12932. doi:10.1038/ncomms12932

Bankhead P, Loughrey MB, Fernández JA, et al. QuPath: open source software for digital pathology image analysis. Sci Rep. 2017;7:16878. doi:10.1038/s41598-017-17204-5

Restructuring of pancreatic islets and insulin secretion in a postnatal critical window. PLoS One. 2006;1(1):e35. doi:10.1371/journal.pone.0000035

Age-related pancreatic tissue postnatal histogenesis in the albino rat (Rattus norvegicus): a morphological, morphometrical and immunohistochemical study with emphasis on alpha and beta cells. Iraqi J Vet Sci. 2024.

Percie du Sert N, Hurst V, Ahluwalia A, et al. The ARRIVE guidelines 2.0: updated guidelines for reporting animal research. PLoS Biol. 2020;18(7):e3000410. doi:10.1371/journal.pbio.3000410

Iwata H, et al. Evaluation of the morphological changes of gastric mucosa induced by a low concentration of acetic acid using a rat model. J Forensic Leg Med. 2014;22:39–44. doi:10.1016/j.jflm.2013.11.007

Dong S, Wang X, Liu Y, et al. A novel gastric ulcer model in rats using filter paper with acetic acid. PLoS One. 2025;20(4):e0319096. doi:10.1371/journal.pone.0319096

Protective effects of melatonin against severe burn-induced distant organ injury: a systematic review and meta-analysis of experimental studies. Antioxidants (Basel). 2020;9(12):1265