Zigmantaitė, Vilma

Background/Objectives: Furan derivatives are commonly encountered in food and environmental matrices and may exert biological effects, but their acute cardiovascular actions and potential mitochondrial targets remain insufficiently characterised. This study examined the effects of two simple furan compounds, 2-acetylfuran (2AF) and 5-methylfurfural (5MFF), on arterial blood pressure in vivo and on oxidative phosphorylation in isolated rat liver mitochondria. Methods: Arterial blood pressure was recorded invasively in anaesthetised rats after intraperitoneal administration of 2AF or 5MFF (0.3 µL/g). Systolic, diastolic, and mean arterial pressures, as well as heart rate, were monitored over time. Mitochondrial respiration was assessed in isolated rat liver mitochondria using high-resolution respirometry. Results: Both 2AF and 5MFF induced a rapid hypotensive response, with significant reductions in systolic, diastolic, and mean arterial pressures within 10–15 min after administration. MAP was reduced to a similar extent by both compounds. However, their chronotropic and pulse pressure responses differed: 5MFF increased heart rate and pulse pressure, whereas 2AF induced a delayed bradycardic response without a significant change in pulse pressure. In isolated liver mitochondria, both compounds markedly reduced ADP-stimulated respiration and decreased the respiratory control index, indicating reduced coupling efficiency. Both compounds also increased the cytochrome c effect, suggesting partial impairment of outer mitochondrial membrane integrity. Conclusions: 2AF and 5MFF exert acute hypotensive effects in anaesthetised rats and impair oxidative phosphorylation in isolated rat liver mitochondria. This study provides the first in vivo evidence that 2AF and 5MFF exert hypotensive effects and identifies them as bioactive furan compounds with dual haemodynamic and bioenergetic actions.

Introduction: Animal welfare, handling and performing procedures in animal research changing approach, has gradually changed since last decades in breeding and animal usefacilities¹,². Nowadays, animal emotional welfare and state becomes the most important factor to grow trust in sciences. Measures for ensuring the welfare of laboratory animals are now applied across all species ³,⁴. The use of artificial intelligence (AI) in experimental animal facilities is still a relatively inovative approach; however, promising results are observed ⁵. Aim of this study was to evaluate two different evaluators and AI for facial expressions scoring. Methods: In the analysis, a generative AI-model (ChatGPT 5.2) was used to assess the same set of rabbit (n=55) facial images for potential signs of pain. AI was provided with a standardized prompt describing rabbit facial pain indicators and asked to classify each image using the same three-point ordinal scale applied for human assessors as well. AI-generated scores were compared with human ratings using weighted agreement statistics. Results: A total of 55 rabbit facial images were independently scored by three evaluators using a three-point welfare scoring scale. Overall inter-rater agreement among the three evaluators was moderate, as indicated by Fleiss’ kappa (κ = 0.55). Friedman’s test revealed no statistically significant differences in overall scoring tendencies between the veterinarian, animal caretaker, and AI evaluator (χ²(2) = 4.47, p > 0.05). This suggests that, at the group level, none of the evaluators consistently assigned higher or lower welfare scores across images. Study results reflect a reasonable level of agreement beyond facial-expression-based welfare assessment across evaluators with different backgrounds. Conclusion: AI–based analysis of animal facial images showed an objective and valid approach to assessing pain and distress. Overall use of AI enhances animal welfare monitoring, reduces human observer bias, and directly supports refinement within the 3Rs principle.

Background/Objectives: Cutaneous squamous cell carcinoma (cSCC) develops through inflammation-driven preneoplastic alterations characterized by epidermal hyperplasia, dysplasia, and increased proliferative activity. C-phycocyanin (C-PC) possesses antioxidant and anti-inflammatory properties; however, its topical potential to attenuate a tumour-promoting cutaneous microenvironment is limited by poor skin penetration. This study evaluated the effects of C-PC-loaded transfersomes in a 7,12-dimethylbenz[a]anthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA)-induced mouse model of skin carcinogenesis. Methods: Male BALB/c mice were assigned to six groups (n = 10 per group). Carcinogenesis was initiated with a single topical application of DMBA, followed by twice-weekly TPA application for 16 weeks. C-PC-loaded transfersomes (1 mg/mL or 10 mg/mL) were applied topically. Histopathological assessment included epidermal thickness, rete ridge depth, mitotic activity, mast cell density, and semi-quantitative scoring of hyperplasia, dysplasia, and inflammation. Ki-67 immunohistochemistry was used to evaluate basal and suprabasal proliferation. Results: Carcinogen exposure induced marked epidermal thickening, severe dysplasia, increased mitotic activity, elevated Ki-67 expression, and pronounced dermal inflammation. Treatment with C-PC-loaded transfersomes significantly reduced epidermal thickness, rete ridge depth, mast cell density, mitotic counts, and suprabasal Ki-67 index. The 1 mg/mL concentration demonstrated the most consistent attenuation of dysplasia severity and inflammatory changes. No adverse histopathological alterations were observed in internal organs. Conclusions: These findings indicate that transfersome-mediated topical delivery of C-PC attenuates early inflammation-driven epidermal remodelling and tumour-promoting alterations in experimental skin carcinogenesis, supporting its potential as a topical preventive strategy.

Pulse Electric Field (PEF) ablation is gaining significant clinical interest as a non-thermal alternative to traditional radiofrequency or cryotherapy-based catheter ablation for the treatment of cardiac arrhythmias. Despite PEF offering improved tissue selectivity and reduced collateral damage, reliable functional validation of lesion depth and electrical effectiveness remains essential for controlling clinical outcomes. We introduce a multimodal cross-validated approach for functional assessment of PEF-induced ablation zones by combining high-speed optical mapping (OM) with microelectrode (ME) transverse and transmural action potential (AP) recordings (Fig.1). Experiments were conducted in a Langendorff-perfused rabbit heart with synchronized PEF delivery, cardiac pacing, and OM acquisition. Critically, lesion depth and electrical impairment were independently verified by recording APs in a cut-off ventricular wedge prepared from PEF-treated regions, enabling direct crossvalidation of surface and transmural electrophysiological changes. Both single-pulse and complex PEF protocols were evaluated. The results indicate that PEF significantly affects lesion size, depth, and functional severity. It slows AP propagation, causes conduction block, and alters AP morphology in peri-ablated tissue. Regions without electrical activity were identified at depths of up to 3 mm, depending on the stimulation parameters used. Overall, OM and ME techniques are useful for investigating and evaluating PEF ablation effectiveness.

Electroporation (EP) is a promising technique for myocardial tissue modification, but its efficacy and safety depend on appropriate parameter selection, limited by the lack of practical methods for three-dimensional (3D) lesion assessment. This study aimed to establish a reliable framework for evaluating EP-induced myocardial lesions and identifying optimal EP parameters. Two complementary approaches were employed. Microelectrode recordings at multiple sites within the EP-treated region determined transmural lesion depth by identifying where action potentials (AP) reappeared, and data from multiple experiments were integrated with AI-based analysis to reconstruct 3D lesion geometry. Lesion size was also assessed using optical mapping by analyzing optical action potential (OAP) amplitudes with depth-weighted exponential changes, and validated by ventricular wall sectioning and direct observation of AP propagation in the wedge. Using near-infrared di-4-ANBDQBS fluorescent dyes (depth constant around 2.1 mm) enabled signal detection and 3D assessment of lesion zones. Both methods enabled precise characterization of lesion dimensions, and optical mapping amplitude analysis (Figure 1A) reliably delineated lesion boundaries and closely matched microelectrodederived transmural profiles (Figure 1B), providing faster and more reproducible evaluation. These findings demonstrate that OAP amplitude measurements can be routinely applied to assess EP-induced lesion 3D zones and guide the optimization of EP parameters for effective and reproducible myocardial EP.

Arterial stiffness, recognized as a marker of vascular aging, may also be influenced by various cardioactive drugs. However, the relationship between blood pressure and stiffness caused by different drugs has not yet been thoroughly characterized. In anesthetized healthy pigs, the acute hemodynamic effects of nine vasoactive substances were systematically evaluated (Fig.1). A single intravenous bolus of each drug was administered via the ear vein, with measurements taken at peak, 5, and 30 minutes post-injection (n=3-5). Real-time measurements of systolic, diastolic, and mean arterial pressure (MAP), pulse pressure, heart rate, and carotid-femoral pulse wave velocity (PWV) were recorded and normalized to baseline. PWV was further normalized to MAP (PWV/MAP) to enable intergroup comparisons of the drug effects, revealing how the induced changes in blood pressure depend on vascular elasticity. All compounds produced distinct, reproducible short-term hemodynamic profiles. α1 and β1 adrenergic stimulation increased PWV, whereas vasodilators and a β2-agonist decreased it. Regression modelling enabled us to evaluate the relationship between blood pressure and stiffness under different drug administrations. This study shows that arterial stiffness is dynamically regulated. We analysed how substances affecting hemodynamic properties can regulate blood pressure and vascular elasticity.

Background: Early tumor-promoting events in skin carcinogenesis are characterized by increased keratinocyte proliferation, epidermal hyperplasia, and architectural alterations of the skin. Natural bioactive compounds with antiproliferative properties may represent promising chemopreventive strategies. C-phycocyanin (C-PC), a cyanobacteria - derived biliprotein, has demonstrated cytostatic and anti-inflammatory effects in experimental models (1-2). However, its topical delivery and influence on early tumor promotion require further investigation. Aim(s): To evaluate whether C-PC-loaded transfersomes modulate early proliferation- and dysplasiaassociated alterations in a mouse DMBA/TPA model of skin tumor promotion. Methods: Experimental skin carcinogenesis was induced using a classical DMBA/TPA initiation– promotion protocol. Animals were treated with C-PC-loaded transfersomes, empty transfersomes, or cancerogenic formulations. Histological assessment included epidermal thickness, rete ridge depth, and dysplasia grading. Immunohistochemical analysis of Ki-67 expression was performed separately in basal and suprabasal epidermal layers. Correlation analysis between suprabasal Ki-67 index and histopathological parameters was conducted. Statistical analysis included Welch’s ANOVA with Games–Howell post hoc test, Kruskal–Wallis analysis, and Spearman correlation (p < 0.05). Results: Tumor promotion induced marked epidermal hyperplasia, increased rete ridge formation, and elevated Ki-67 expression extending into suprabasal layers. Treatment with C-PC-loaded transfersomes significantly reduced epidermal thickness and suprabasal proliferative activity compared to untreated promoted skin. Suprabasal Ki-67 index positively correlated with epidermal thickness, rete ridge depth, and dysplasia grade, indicating its sensitivity as a marker of early pathological proliferation. Conclusion: Topical delivery of C-PC via transfersomes was associated with attenuation of early proliferative and structural alterations during skin tumor promotion in this experimental model. Further studies are required to clarify the underlying mechanisms and to determine their potential relevance in long-term chemopreventive strategies.

Introduction Around 15% of cases of acute kidney injury (AKI) progress to chronic kidney disease (CKD). Although human placenta-derived mesenchymal stromal cells (hpMSCs) demonstrate anti-inflammatory properties, their long-term effects are unclear. This study aimed to evaluate the preventive effect of hpMSCs on AKI-to-CKD progression and to assess biomarkers sensitivity and correlation. Methods hpMSCs were isolated, cultured, and characterised by yield, viability, phenotype and immunosuppressive potency. A rat model of ischaemia-reperfusion injury (IRI): both renal pedicles were clamped for 60 minutes. The cells group (IRI-hpMSCs) received 3×105 hpMSCs in each kidney. Control groups received phosphate buffer solution (PBS), IRI only, or no intervention (healthy). Blood (creatinine), urine (KIM-1 and cystatin C) and kidney samples were collected on days 0, 3, 7, 21–28, 56 (n ≥ 3 per timepoint; total n = 57). Serum creatinine was measured in surviving animals. Kidney histology included scoring of acute tubular necrosis (ATN), tubular dilatation (TD), casts, loss of brush border (LBB), and interstitial fibrosis and tubular atrophy (IFTA), with injury quantified by renal injury score (RIS). Calculated correlations. Statistical analysis was performed using SPSS 17.0 and GraphPad Prism 7.04 with non-parametric tests (p < 0.05) and survival curve analysis. Results hpMSCs showed consistent viability, expressed MSC markers, and dose-dependently suppressed T-cell proliferation. hpMSCs significantly improved kidney function, with a lower median of serum creatinine levels on day 7 (vs. IRI p = 0.014; vs. PBS p = 0.034). Cystatin C levels were reduced on day 3, and KIM-1 levels were lower on day 14 vs. PBS in the cells group. Histology revealed that hpMSCs reduced renal injury in both early and late phases. On day 3, the cells group showed significantly less ATN, reduced cast formation, and absence of focal coagulative necrosis by day 7. On days 21–28, RIS, TD, LBB, cast formation, and IFTA were significantly lower in the cells group compared to both controls. IFTA was significantly lower in the cells group on day 56 (vs. IRI, p = 0.018; vs. PBS, p = 0.049). Serum creatinine normalized on day 28 and 56, but fibrosis persisted only in controls. Despite the limited sample size, several strong correlations were found between early urinary biomarkers and later histological IFTA scores. In the IRI-PBS group, higher KIM-1 (day 14) and cystatin C (day 3) levels were significantly associated with increased IFTA at day 28, but inversely correlated with IFTA at day 56, suggesting possible prolonged kidney damage or delayed regeneration. An increase in cystatin C on day 3 is strongly associated with an increase in IFTA on day 56 in the IRI group, statistically significant. Notably, in the placental cell group, cystatin C elevation (day 3) showed a strong inverse correlation with IFTA at day 28, indicating potential early regenerative effects. At the end of the study, 100% of the rats in the cells group survived, compared to 68% in the PBS group (p = 0.010) and 72% in the IRI group (p = 0.021).

Background and Aims

Recurrent episodes of acute kidney injury (AKI) progress to chronic kidney disease (CKD) in 15% of patients. Allogeneic type human placental mesenchymal stromal cells (hpMSCs) have been shown to have short-to-medium term beneficial effect in preventing the progression of CKD; however, long-term efficacy and immunogenicity remains unknown. We aimed to assess the preventive effect of hpMSCs on renal fibrosis after preclinical ischaemia-reperfusion injury (IRI) and to prevent the development of CKD.

Method

HpMSCs was isolated using Good manufacturing practice (GMP) grade materials, cultivated and characterised by yield, viability, flow cytometry and in vitro potency for immunosuppression. Rats underwent preclinical IRI - both kidney roots were clamped with atraumatic microvascular clamps and were removed after 60 minutes of occlusion. After removal of the clamps, the IRI-hpMSC treated group (cells group) received 3×10⁵ hpMSCs into the corticomedullary region of each kidney. IRI-PBS control group (PBS group) received phosphate buffer solution (PBS); untreated group with only induced IRI (IRI group); healthy control group without IRI. Survival analysis was carried out till day 56. Blood and kidney tissue samples collected on the days 0, 3, 7, 21–28, and 56. At least 3 rats per end point (a total of 57 rats). Serum creatinine was analysed in surviving animals. Kidney histology. Acute tubular necrosis (ATN), dilatation (TD), casts, loss of brush border (LBB), tubulitis, leukostasis, interstitial fibrosis and tubular atrophy (IFTA) were scored in 10 fields of each section. The degree of renal injury was assessed by assessing the percentage of affected tubules - renal injury score (RIS). Statistical analyses were performed using SPSS Statistics version 17.0 and GraphPad Prism 7.04 software. Non-parametric data were analyzed using the median test and Kruskal-Wallis Test (statistical significance p-value < 0.05). Survival - the curve comparison test.

Results

hpMSCs had a consistent yield, viability, MSC expression markers and suppressed proliferation of T cells in a dose-dependent fashion. Rats survived 100 percent in cells group while only 68 percent in PBS (P = 0.010) and 72 percent in IRI group (P = 0.021), at the end of the study. hpMSCs significantly improved kidney function by decreasing median creatinine level in serum and on day 7 compared to the surviving rats in comparison to the death-censored control groups (Cells vs IRI P = 0.014; Cells vs PBS P = 0.034). The histological findings indicated that hpMSCs mitigated the injury to renal morphology in both the early and especially at the late phases. The cells group exhibited significantly lower median level of ATN on day 3 compared to the IRI (P = 0.047) and PBS (P = 0.028) groups. Comparatively, the cells group had a distinct tendency toward a smaller casts formation (Cells vs IRI P = 0.014; PBS P = 0.034), with ATN devoid of focal coagulative necrosis visible as early as day 7. On days 21–28, significant RIS differences were observed between cells group and both IRI (P = 0.048) and PBS (P = 0.029) groups. TB (Cells vs IRI P = 0.019; PBS P = 0.017), LBB (Cells vs IRI P = 0.048; PBS P = 0.017), casts formation (Cells vs IRI P = 0.048), and IFTA (Cells vs IRI P = 0.048; PBS P = 0.029) were significantly lower in the cells group than in the IRI and PBS groups on days 21–28. IFTA was significantly lower in the cells group than in the IRI (P = 0.018) and PBS (P = 0.049) groups on day 56. Serum creatinine normalized on day 28 and 56, but fibrosis persisted only in controls.

Conclusion

Perinatal stem cells have the potential to prevent acute kidney damage and consequential fibrosis cascade. The results of the histological study demonstrated that perinatal stem cells provided long-lasting (56 days) protection against fibrotic kidney injury. A superior 100% survival rate of treated animal group exhibits the potential of characterised hpMSCs to be used in a larger scale preclinical tumorigenicity and toxicity studies and subsequent clinical studies.

Stents are medical devices used to treat the narrowing of the blood vessel, most commonly caused by atherosclerosis. Currently used bare-metal, drug-eluting stents are limited in size and architectural complexity, and there are a few risks associated with these medical devices. In some cases stents can cause thrombosis or even death. Furthermore, said risk increases while stenting relatively small vessels. This paper shows that vascular stents for relatively small vessels and/or for patient-specific stenting applications can be printed using two-photon polymerization (2PP). 2PP is an additive manufacturing technique with sub-μm resolution and unlimited 3D geometry potential. These capabilities were used to produce stents for small blood vessels, which are 5 mm tall and 0.7 or 0.9 mm in diameter, with 3D struts as thin as 50 μm. Several novel approaches were introduced to accommodate the printing of such a structure like voxel elongation and printing in stereolithography-like vat-sample holder configuration. Furthermore, the produced stents were tested mechanically proving their mechanical resilience to most common types of mechanical deformations. Experimental results were also compared to mathematical modeling, showing excellent agreement, hinting at the possibility of designing and testing complex micro-stent geometries before fabrication in silico. Finally, biocompatibility experiments were performed, in which rats survived the 7-day incubation period and showed no significant biocompatibility issues. Overall, the presented work gives an outlook on all aspects related to the 3D printing of stents - design, manufacturing, mechanical, and biological testing. We show that 2PP aligns very well with all these aspects and is a promising technique for the mass manufacturing of vascular stents for small vessels or specifically for the patient.