Diagnosing fatal hypothermia remains a forensic challenge due to the lack of specific postmortem markers. This study investigates the potential of extracellular signal-regulated kinase 1 (ERK) and atrial natriuretic peptide (ANP) as biochemical indicators in hypothermia-related deaths, particularly under the influence of ethanol and diazepam. A randomized experimental study was conducted in rats approximately 3.5 months old and weighing 200–240 grams, which were randomized into the control group (n=7), the ethanol-treated group (n=7), and the diazepam-treated group (n=7). Survival time was measured from the point of immersion in 7°C water until the animals’ death. Hypothermia was induced, and serum and muscle levels of ERK1 and ANP were measured using ELISA. Serum ERK1 levels differed significantly across groups (p = 0.039), with a similar changes in Control and Ethanol groups, while Diazepam decreased. ANP levels showed no significant changes across groups or time points. Muscle concentrations of both markers remained stable. The observed alterations in ERK1 levels may indicate a possible association with stress responses during hypothermia, although the findings should be considered exploratory due to limited statistical significance after multiple-comparison correction. ANP showed limited diagnostic utility under the study conditions. Further research incorporating histological validation and larger cohorts is needed to confirm these findings and improve forensic applications.

Background/Objectives: Establishing drowning as the cause of death remains one of the most challenging tasks in forensic pathology, particularly when classical autopsy findings are absent, nonspecific, or compromised by decomposition. Molecular biomarkers may provide objective adjunctive evidence in such cases. This study investigated the diagnostic utility of Aquaporin-5 (AQP5) and Surfactant Protein A2 (SP-A2) measured in renal tissue as potential biomarkers of freshwater drowning and evaluated the influence of different freshwater environments on their expression. Methods: A controlled experimental study was performed using 29 adult Wistar rats allocated to freshwater drowning groups (Boračko Lake, n = 11; Jablaničko Lake, n = 11) and non-drowning control groups subjected to mechanical asphyxia followed by one hour of postmortem immersion (n = 7). Kidney tissues were collected within a maximum of 5 min after death and analyzed for AQP5 and SP-A2 concentrations using enzyme-linked immunosorbent assay (ELISA). Group differences were evaluated using Kruskal–Wallis and Dunn’s post hoc tests, while effect sizes were estimated using Cliff’s delta. Receiver operating characteristic (ROC) curve analysis was performed to assess the diagnostic performance of each biomarker in distinguishing drowning from non-drowning deaths. Results: AQP5 concentrations differed significantly among groups (χ2 = 16.05, p = 0.00033), with large effect sizes observed in all pairwise comparisons. However, diagnostic performance was poor, as ROC analysis demonstrated no significant discrimination between drowning and non-drowning cases (AUC = 0.534, 95% CI: 0.314–0.754; p = 0.762). In contrast, SP-A2 showed significant intergroup differences (χ2 = 6.69, p = 0.035) and moderate diagnostic accuracy for drowning identification (AUC = 0.752, 95% CI: 0.567–0.937; p = 0.008), with a sensitivity of 77.2% and specificity of 81.8% at the optimal threshold. Notably, AQP5 expression appeared suggestive of environmental modulation, whereas SP-A2 demonstrated greater relevance to the drowning process itself. Conclusions: Renal SP-A2 showed moderate diagnostic utility as a supportive biomarker for drowning, whereas AQP5 lacked sufficient discriminatory power for cause-of-death determination. The marked variability of AQP5 across freshwater environments suggests that it reflects environmental modulation rather than drowning-specific biological responses. Although neither marker is suitable as a standalone diagnostic tool, renal SP-A2 may represent a useful adjunct in forensic drowning investigations, particularly when pulmonary tissue is unavailable or unsuitable for analysis. These findings should be considered preliminary and applicable only to freshwater drowning conditions, as saltwater drowning was not investigated in the present study. Further validation in human forensic casework and larger experimental cohorts is warranted.

Rats are opportunistic omnivores, consuming both plant- and animal-derived foods, and therefore represent a suitable animal model for investigating the potential adverse effects of heterogeneous diets on hematological and biochemical blood profiles. The study included 18 Wistar rats of both sexes, aged 1–2 months, randomly allocated into three groups of six animals each. For a period of seven weeks, Group H was fed bakery products, Group M received meat and meat products, while the control Group K was fed a conventional pelleted diet. Biochemical parameters were analyzed from peripheral blood samples collected from the experimental animals. Statistically significant differences (p < 0.05) were identified among the groups for CREA, UREA, PHOS, Ca, TP, ALB, GLOB, ALB/GLOB, CHOL, and LIPA. No significant differences were observed for the remaining parameters. The biochemical findings indicate impaired renal function in rats from the meat-fed group, as evidenced by elevated creatinine, urea, phosphorus, and lipase levels. In addition, reduced concentrations of serum proteins, particularly albumin, were observed across all experimental groups, suggesting inadequate protein synthesis resulting from insufficient intake of essential amino acids and micronutrients required for normal liver function.

The aim of this study was to assess the possible adverse effects of different dietary regimens—namely diets based onbakery products and meat products—on erythrocytes, their morphology, erythrocyte indices, platelets, leukocytes, andthe leukogram in rats during late puerperium. A monotonous diet may affect the hematological profile and potentiallycause pathophysiological disorders. The study included 18 clinically healthy rats in the late puerperal period, divided intothree groups of six animals each: a group fed meat products (Group M), a group fed bakery products (Group H), and acontrol group (Group K) that consumed standard pelleted rodent feed. All groups of rats were fed their respective dietsseparately for a period of 49 days. At the end of the experimental period, hematological blood analyses were performed.Statistically significant differences (p < 0.05) were found among the groups for the parameters MCV, MCH, and PLT. It wasalso established that HCT, HGB, MCV, and MCH values in the experimental bakery-product group (H) were lower than boththe physiological reference intervals and the corresponding values in the control group (K). Moderate anulocytosis wasobserved, accompanied by hypochromic erythrocytes of reduced volume. Regarding the experimental group that consumedprocessed meat products, the results indicated the presence of hypochromic anemia, as evidenced by decreasedHGB, MCV, and MCH values. Examination of peripheral blood smears revealed the presence of poikilocytotic erythrocyteforms, namely echinocytes, with a notable percentage occurrence (14.05%) within microscopic fields of view. A diet basedon bakery products led to changes in hematological parameters, resulting in the development of iron-deficiency anemia.Furthermore, prolonged consumption of meat products contributed to the development of hypochromic anemia and suggestedpossible impairment of kidney function. This study demonstrated that rats during the late puerperal period are notimmune to the adverse effects of a monotonous meat-based diet nor to a monotonous diet consisting of bakery products.

A proper diet that provides balanced nutrients according to species, breed, age, sex, and purpose is essential for achieving optimal animal growth, development, and physiological function. In relation to this statement, the aim of this study was to quantitatively assess the effects of different dietary regimens on the morphometric characteristics of the small intestine of late puerperal rats, with a emphasis on the duodenum and jejunum. The research included measurements of the height and width of intestinal villi, and the depth of crypts, in order to determine specific morphological changes associated with the influence of the diet. The experiment involved 18 adult rats divided into three groups: the first group received standard commercial rat feed (control group), the second group was fed bakery products, and the third group was given a diet consisting exclusively of meat. Over a 49-day period, body weight, organ weights, and intestinal segment lengths were measured. Histological analyses of duodenum and jejunum samples were performed, alongside detailed morphometric assessment of the intestinal mucosa. Significant alterations in intestinal villi were observed. In the duodenum, the greatest villus height and width, as well as crypt depth, were observed in rats that had meat-based diet. Similarly, in the jejunum, rats from the same group exhibited the greatest villus height and crypt depth, while the mean measure of the villus width was almost identical to mean measure in rats that were fed with commercial food. These findings suggest that diet composition profoundly influences intestinal architecture and function. Overall, the study emphasizes the critical role of balanced nutrition in maintaining gastrointestinal health, systemic homeostasis, and optimal development, not only in laboratory animals but also in a broader veterinary and biomedical context.

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by inflammation and destruction of cartilage, as well as by extra-articular manifestations. Rheumatoid nephropathy is a common complication of RA and its principal target is the renal corpuscle. Vitamin D and its analogs exert immunomodulatory actions throughout the body due to the widespread of their receptors. Our study aimed to compare the effects of cholecalciferol (vitamin D3) and alfacalcidol on renal corpuscle changes in pristane-induced RA model following a 28-day treatment, using geometric morphometrics. Forty female Wistar rats (190–210 g; 12–13 weeks old) were randomly assigned to four groups: the control (Cont) group (n = 10) received saline i.c., the PIA group (n = 10) was administered pristane i.c., PIA-ALF group (n = 10) was administered pristane i.c. and alfacalcidol orally, and the PIA-CH group (n = 10) was injected i.c. with pristane and received cholecalciferol orally. Pristane administration was used for RA induction. At the end of the experiment, the left kidneys were removed and processed by standard histological procedures for geometric morphometric analysis. Geometric morphometric analysis demonstrated that, compared with the control group, the architecture of the renal corpuscles was altered in the PIA (p < 0.0001) and PIA-CH (p = 0.0065) groups. In contrast, no statistically significant differences were observed in the PIA-ALF group (p = 0.3011). Geometric morphometric analysis demonstrated that alfacalcidol, but not cholecalciferol, exertedaprotective effect on the renal corpuscle architecture in pristane-induced rheumatoid arthritis in rats.

Abstract.

Background: Many deaths during heat waves stem not only from body overheating but also from heat stress, which can intensify pre-existing medical conditions, leading to fatal outcomes. Aim: This study aimed to investigate whether the intensity of pathological changes in the heart muscle and lung tissue of albino rats exposed to hyperthermia correlates with different water temperatures (WT) and to determine whether the histological structure of the myocardium and lungs varies. Methods: A sample of 21 albino rats was exposed to water temperatures of 37°C, 41°C, and 44°C. Temperature readings were recorded before immersion, immediately after immersion, at the point of reaching hyperthermia, at 20 minutes, and at the time of death. Tissue samples were collected from the dissected rats, fixed in 10% buffered formalin at room temperature, embedded in paraffin, sectioned into 4–5 μm slices, and stained using the hematoxylin-eosin method. Results: The severity of myocardial histopathological alterations increased with both higher temperatures and longer exposure durations WT. However, the progression of morphological alterations in cardiomyocytes was not markedly significant, likely due to the brief exposure time, which limited the visualization of subcellular alterations in Hematoxylin and Eosin staining-stained tissue. All lung samples from the seven rats exposed to the highest temperatures displayed bronchiolitis and acute bronchitis, along with early bronchopneumonia. Conclusion: While some organs exhibit greater tolerance to heat stroke than other organs, most organs show similar alterations characterized by capillary dilation, vascular pathway disruption, and extravasation. The extent of pathological changes in myocardial and lung tissue intensified with higher temperatures and longer exposure durations to elevated WT. However, the progression of morphological alterations in cardiomyocytes did not demonstrate marked significance, likely because of the brief exposure period. This short duration may limit the detection of subcellular changes when using hematoxylin-eosin staining.

Background: Primary hypothermia occurs when the body is exposed to extremely low temperatures in an environment with no underlying health conditions. Secondary hypothermia, on the other hand, results from disruptions in thermoregulation due to diseases, trauma, surgery, drugs, alcohol, or infections. Postmortem biochemistry has become a crucial factor in forensic examinations, offering valuable apprehension into tissue of and organ dysfunction associated with the process of dying. Aim: This research aims to explore various biochemical markers and their significance in distinguishing primary from secondary hypothermia. Methods: This study involved 21 Wistar rats, which were separated into three experimental groups: CG (n = 7), which were exposed only to hypothermic conditions; AHG (n = 7); and BHG (n = 7). We tested these parameters in each rat: glucose, urea, creatinine, blood urea nitrogen to creatinine ratio, phosphorus, calcium, sodium, potassium, sodium to potassium ratio, chloride, and calculated osmolality. Results: Distinct biochemical differences were noted between primary and secondary hypothermia. Glucose and creatinine levels exhibited significant variations (p < 0.001). Urea concentrations also manifested notable differences between the groups (p < 0.001). Phosphorus levels demonstrated significant differences (p = 0.004), with post hoc analyses revealing significant contrasts between the AHG and BHG (p = 0.014) and between the BHG and CG (p = 0.014). Potassium levels and the sodium-to-potassium ratio differed significantly (p < 0.001). Osmolality also varied significantly across experimental groups (p < 0.001), with post hoc tests confirming significant differences between the AHG and CG (p = 0.013) and between the BHG and CG (p = 0.002). Conclusion: The calculated osmolality exhibited significant variation among the different groups, indicating a notable impact of the substances on the biochemical profile related to hypothermia. This study focused on the effectiveness of biochemical markers in distinguishing primary hypothermia from secondary hypothermia.