Animals: 54 Wistar SPF rats (50% female) of 8 weeks with weights of 200 +/- 10 grams from the University of Antioquia biotherium were used. The rats (our experimental unit) were, housed with a maximum density of 3 animals per box, with access to concentrated feed and water at will, temperature between 20°C and 25°C, under controlled light-dark conditions. Ethical aspects: the study was approved by the Animal Experimentation Committee of the University of Antioquia (1-2025).
Animal model of toxicity: in the laboratory, all animals received 30 mg/kg of paraquat ion by oral probe, this dose is within the range of lethal dose 50 described for rats (http://pmep.cce.cornell.edu/profiles/extoxnet/metiram-propoxur/paraquat-ext.html#10). We estimated a sample size of 9 rats per group to detect minimum differences of 50% in the degree of acute inflammation between the placebo group and each of the experimental groups, assuming an alpha error of 5% and a power of 80 %.After 2 hours post-intoxication, groups of 9 animals were randomly assigned (using a sequence of random numbers generated by STATA ®) in one of the following six experimental arms: (1) cyclophosphamide 15 mg/kg plus dexamethasone 30 mg/kg (single doses), (2) low molecular weight heparin 60, 80 or 100 U/kg every 12 h subcutaneously for two days (3 animals per dose) , (3) unfractionated heparin (UFH) 5, 25 or 50 mg/kg every 24 h intratracheal (3 animals per dose) for two days , (4) vitamin C 20, 40 or 60 mg/kg every 24 h (3 animals per dose) for three days , (v) atorvastatin 10, 20, or 40 mg/kg every 24 h (3 animals per dose) for eight days , or (vi) placebo with intraperitoneal saline (single doses). Health conditions were evaluated every three hours for up to 21 days while the rats survived. All experimental groups were treated and assessed at the same time.
Histopathological analysis: at the end of the treatment, euthanasia for cervical dislocation was performed under anesthesia with isoflurane (Abbott, USA). All evaluators were blinded to the assigned treatment. The extracted tissues were placed in a 10% formalin solution and embedded in paraffin to make sections with the 4-micron microtome. The tissues were initially colored with hematoxylin and eosin for evaluation under conventional optical microscopy. In the lung, Masson's trichrome staining samples were performed to quantify collagen deposition. In the liver, trichrome staining, PAS, PAS diastase and cytokeratin 7 were used to assess bile duct integrity and CD68 to quantify Kupffer cells. In the kidney, trichrome stain, kidney methenamine silver, Masson fontanel, and PAS were used to evaluate the glomerular basement membrane and quantify the presence of hyaline globules in renal tubules. The histopathological evaluation of all the samples was carried out by a group of three pathologists and two pathology residents, when there was disagreement in any of the cases, the concept of an additional expert was requested.
Histopathological evaluation in lung: lung inflammation was evaluated in the interstice and airway. The degree of interstitial inflammation (presence inflammatory infiltrate) was classified in three categories: mild inflammation (inflammatory compromise <25% in the sample analyzed), moderate (inflammatory compromise of 25 to 50% in the sample analyzed), and severe (inflammatory compromise >50% in the sample analyzed). The degree of alveolar injury was evaluated in three categories: severe alveolar injury was considered if there was the destruction of alveolar architecture with hemorrhage and edema. The moderate alveolar injury was considered if there were only one of these two findings, and mild alveolar injury was considered when alveolar architecture was preserved and there was absence of hemorrhage and edema. Interstitial fibrosis was evaluated under the Aschroft scale. A normal lung or minimal wall thickening without architectural damage was considered mild fibrosis. Moderate wall thickening of defined lung structure or fibrosis with defined lung structure damage were considered as moderate fibrosis, and severe distortion of fibrotic structures or total fibrous obliteration were considered as severe fibrosis.
Histopathological evaluation in liver and kidney: histopathological changes in liver and kidney were evaluated in 33 rats. The hepatocyte damage was evaluated according to the presence or absence of liquefactive or ischemic necrosis foci. To classify hepatic regeneration the variables of ballooning, binucleation and increased mitosis in hepatocytes were evaluated. With three characteristics fulfilled it was considered as severe regeneration if it presented only two were considered as moderate regeneration and mild with only one of them. Other histopathological variables evaluated were ductopenia, stage of destruction of the ductal epithelium, increase of Kupffer cells, fibrosis and congestion. In the kidneys, acute tubular necrosis and kidney congestion were evaluated qualitatively as present or absent.
Statistical analysis: The unit of analysis was a single animal. Fisher's exact test was used to compare frequencies of number of rats by histopathological findings between treatments. Values of p less than or equal to 0.05 were considered as statistical significance in the hypothesis tests.