Motility is a fundamental property of microglial processes. Their dynamic movement is highly sensitive to alterations in the surrounding microenvironment and serves as an early indicator of pathological conditions in the central nervous system (CNS). Thus, comprehensive and accurate quantification of microglial process dynamics is critically needed. To address this, we developed a novel approach based on 3D alignment of original two-photon time-lapse images to correct misalignments caused by microglial migration or animal motion. This alignment facilitates subsequent colocalization analysis, allowing quantification of complete microglial structural dynamics at single-pixel resolution. Compared to conventional methods, our technique offers enhanced comprehensiveness and precision, enabling simultaneous measurement of process extension or retraction, microglial loss or regeneration, and clear visualization of temporal structural changes. Finally, we quantified the dynamic motility of microglia in abilateral common carotid artery occlusion/reperfusion (BCCAO/R) model using this method. We anticipate that this method will significantly improve the quantitative accuracy of dynamic motility assessments for microglia and other cell types in vivo.

Liver fibrosis is characterized by scar formation as a response to injury. Although many drugs have demonstrated antifibrotic efficacy in vitro and in animal models, effectively translating these into effective clinical therapies remains a challenge. Nanotechnology offers potential therapeutic approaches for treating liver fibrosis. This study aimed to assess and compare the antifibrotic effects of losartan and losartan nanoparticles (LP-NPs) on CCl4-induced liver fibrosis in rats.Thirty-two adult male Sprague-Dawley rats were divided into four groups: a control group, a CCl4-induced fibrosis group, a losartan-treated group, and an LP-NPs-treated group. Liver fibrosis was induced by CCl4 injections over six weeks. Losartan or LP-NPs were orally administered for four weeks. At the conclusion of the study, rats were euthanized, and blood samples were collected for liver enzyme assessment. Liver specimens were subjected to histopathological examination (H&E, Sirius red staining), immunohistochemistry (α-SMA and TIMP1), and quantitative real-time PCR (qRT-PCR) for gene expression analysis of α-SMA and TIMP1. Morphometrical studies quantified collagen areas in Sirius red-stained sections and optical density of α-SMA and TIMP1 in immunohistochemically stained sections.The results showed that CCl4 induced significantly elevated liver enzymes, severe distortion of hepatic architecture, marked steatosis and fibrosis. Additionally, there was an increase in collagen content, accompanied by strong positive immunostaining and upregulated gene expression of α-SMA and TIMP1. Both losartan and LP-NPs treatments significantly improved these parameters compared to the CCl4-only group. Notably, LP-NPs demonstrated significantly greater improvements in liver enzyme levels, collagen percentage area, and the gene expression of α-SMA and TIMP1 compared to the standard losartan group. However, no significant difference was observed between the two treatments in α-SMA optical density.In conclusion, this investigation demonstrates that nanoformulation significantly enhanced losartan's antifibrotic efficacy. LP-NPs improved liver function and histological architecture, reduced collagen content after CCl4 administration, and demonstrated greater therapeutic efficacy than conventional losartan.

In vitro cell culture models are fundamental to biomedical research, yet the choice between primary cell cultures and immortalized cell lines profoundly influences experimental outcomes. Immortalized lines offer convenience and unlimited proliferative capacity but frequently acquire genetic, epigenetic, and phenotypic alterations that compromise tissue-specific functions and overall biological fidelity. In contrast, primary cell cultures preserve native morphology, signaling pathways, and functional characteristics of their tissue of origin, providing a more physiologically relevant model. While previous reviews have outlined the general advantages of primary cells, few critically synthesize comparative data across cell types or evaluate strategies to mitigate inherent limitations such as finite lifespan and donor variability. This review critically examines the comparison between primary and immortalized cells across different cell types in terms of preserved tissue-specific characteristics, genomic and epigenetic fidelity, predictive value in drug testing and disease modeling, and emerging roles in personalized medicine. We further discuss innovative approaches, including conditional reprogramming, optimized donor selection, pooled-donor strategies, and standardized culture protocols, that enhance reproducibility and functional stability. By integrating comparative evidence with methodological advances, this review provides a critical framework for selecting appropriate in vitro models, highlighting the translational significance of primary cells in drug discovery, precision medicine, and biomedical research.

Histologic stains which identify specific tissue and cellular components are a staple of all diagnostic pathology laboratories. Histotechnology scientists are constantly attempting to find methods and reagents which are specific, simple to use, and cost effective. Turmeric (Curcuma longa) is a yellow spice readily available in grocery stores and is inexpensive. Previously, turmeric has been investigated as a natural histological stain, with most studies describing predominantly cytoplasmic staining and suggesting turmeric as a replacement for eosin. However, the present study supports the use of turmeric as a counterstain as staining localization is strongly dependent on the solvent, co-solute and procedure used for extraction and for staining. In contrast to several prior reports using alcoholic turmeric extracts, alcohol-based preparations in this study produced little to no appreciable tissue staining despite exhibiting intense coloration in solution. Instead, an aqueous extract with addition of aluminum was the most effective. A 0.5% turmeric solution prepared in aqueous 5% aluminum sulfate produced a bright yellow stain in histologic sections. While yellow chromatin against a colorless background provided limited contrast as a routine nuclear stain substitute, pairing turmeric with selected companion stains yielded favorable results. As a counterstain for alcian blue, Prussian blue, von Kossa, and rhodanine methods, turmeric provided excellent visual contrast. Turmeric is also markedly less expensive than nuclear fast red, supporting its potential as an economical alternative for selected histological applications.

Coccidiosis is a significant enteric disease impacting economically important livestock, caused by a multidrug-resistant parasite. This research was performed to assess the immunohistochemical and molecular insights of Coriandrum sativum leaves extract (CSLE) against Eimeria papillata infection in murine models. CSLE subjected to phytochemical analysis. Thirty male Swiss albino mice were included: Group 1: Control; Group 2: Supplemented group receiving CSLE at 300 mg/kg B.W. (CSLE); Group 3: Infected with 1000 sporulated E. papillata oocysts; Group 4: Infected-treated with CSLE at 300 mg/kg B.W. (Infected + CSLE). Group 5: Infected-treated with amprolium at 120 mg/kg B.W. (Infected+Amp). Following 60 min of infection, groups 4 and 5 received daily treatment for five successive days with CSLE and Amp, respectively. Measurements of oocyst suppression and jejunal glutathione peroxidase (GPx) activity were conducted. Concentrations of Caspase-3 (Casp3) as well as jejunal nuclear factor kappa B (NFκB) and Casp-3 cells were measured immunohistochemically. Gene expression of apoptotic genes was assessed. The CSLE exhibited a substantial concentration of total phenolics and flavonoids, in addition to notable antioxidant activity (48.04 ± 2.98% DPPH scavenging). CSLE treatment exhibited significant oocyst suppression and minimized parasitic stages in the jejunum. CSLE reinstated jejunal GPx activity and mitigated the infection-induced elevation in Casp3 protein levels and its gene expression. Furthermore, CSLE modified apoptotic gene expressions, significantly downregulating BCL-2-associated protein x (BAX) and upregulating B-cell leukemia/lymphoma 2 (BCL2) following CSLE therapy. These data collectively underscore the significant anticoccidial, antioxidant, and antiapoptotic capabilities of CSLE, indicating its potential as a natural therapeutic treatment for E. papillata infection.

Despite the commercial success of protein- and peptide-based therapeutics, their clinical application remains limited due to rapid clearance and inactivation via metabolic pathways. In this study, we PEGylated Aspergillus flavus-derived uricase (Uox) using methoxy polyethylene glycol (5 kDa) activated with Succinimidyl Succinamide (mPEG-SS) at a molar ratio of 1:10 (Uox:mPEG-SS) to enhance its pharmacological properties. This study aimed to evaluate and compare the kinetic behavior, thermodynamic stability, and immunogenic potential of native and PEGylated uricase. mPEG-SS was synthesized through a two-step reaction and characterized by FT-IR,1 H-NMR spectroscopy, and TLC. Successful conjugation to Uox (mPEG-SS@Uox) was confirmed using DLS and SDS-PAGE. Kinetic and thermodynamic analyses revealed a 16.44% increase in thermal stability and a 23.37% increase in half-life at 45°C upon PEGylation, with only a modest 27.3% reduction in catalytic efficiency, likely due to steric hindrance. Immunogenicity assays using CFSE-labeled PBMC proliferation and cytokine profiling via ELISA indicated that PEGylated Uox induced significantly lower immune activation compared to the native enzyme (p < 0.0045), with no statistical difference from the control group (p = 0.2426). These findings demonstrate that PEGylation with 5 kDa mPEG-SS substantially improves uricase's stability and diminishes its in vitro immunogenic potential (as evidenced by reduced PBMC activation), making it a promising candidate for further preclinical development in the treatment of hyperuricemia and gout.

The Greater Caribbean manatee, recently reclassified as endangered, is a keystone species in coastal and freshwater ecosystems. Its long-term conservation requires integrative strategies that combine in situ and ex situ approaches. Although skin-derived cell cultures have been reported for other sirenian species, none have yet been established and characterized for the Greater Caribbean manatee. Thus, the aim of this study was to establish and characterize skin-derived fibroblast cultures from the Greater Caribbean manatee. Skin biopsies obtained from seven animals were used to establish primary cultures. The cells were expanded in vitro, identified as fibroblasts by vimentin immunostaining, and characterized by ultrastructural features, karyotype stability, and the absence of microbial contamination under antibiotic-free culture conditions. Fibroblasts at passages 2-10 were characterized for morphology, viability, metabolic activity, population doubling time, oxidative stress levels, mitochondrial membrane potential, and apoptotic profile. Explant-derived cultures adhered within 24 h, reached sub-confluence after a mean of 8.4 days, and a full confluence by 17.9 days, sustaining active proliferation for up to 84.6 days across ten passages. Cells displayed typical fibroblast-like morphology, high viability (≥ 84%), and stable metabolic activity and proliferation rates (P > 0.05) throughout the culture period. Reactive oxygen species levels increased significantly at passages 4, 8, and 10 relative to passage 2 (P < 0.05), accompanied by a mild reduction in mitochondrial membrane potential, while overall cellular functionality was preserved. Apoptotic and necrotic events exhibited only transient fluctuations without evidence of progressive cellular damage. Cytogenetic analyses revealed a stable karyotype of 2n = 48, with normal XX/XY constitution and no detectable chromosomal abnormalities. Cultures remained free of microbial contamination for 30 days under antibiotic-free conditions. Therefore, this study establishes the first established fibroblast culture from the Greater Caribbean manatee, offering a novel and reliable platform for functional, genomic, cytogenetic, and toxicological studies in conservation biotechnology.

This study aimed to investigate the changes in HSP27 and HSP70 immunoexpression after hyperthermia in the lung and heart of rats on days 5, 10, 20, 25, and 30 of postnatal development. The body temperature of rat pups in the hyperthermia groups was increased to 40°C under the infrared lamp. The recovery time after hyperthermia was kept at 5 hours. HSP27 was present in cardiomyocytes and bronchiole and alveolar epithelial cells of control rats. Compared to the control groups after hyperthermia, HSP27 expression was only significantly different on day PND10 for cardiomyocyte cells (P < 0.05), but not on any day of postnatal development for bronchiole and alveolar epithelial cells (P > 0.05). In control group rats, HSP70 was almost absent in cardiomyocytes and alveolar epithelial cells but was evident in bronchiole epithelial cells. A significant increase in HSP70 expression after hyperthermia was observed throughout the postnatal days in cardiomyocytes and alveolar epithelial cells, but only on day PND20 in bronchiole epithelial cells (P < 0.05). In conclusion, during postnatal development, HSP27 is essential for the development of cardiomyocytes and bronchiole and alveolar epithelial cells, and HSP70 is essential for the development of bronchiole epithelial cells. On the other hand, HSP70 may participate in cytoprotective and repair mechanisms against hyperthermia-induced stress in cardiomyocytes and alveolar epithelial cells during postnatal development.

Canine mammary gland tumors (CMGTs) are among the most common neoplasms in female dogs, and serve as valuable models for human breast cancer in terms of histopathology and biological behavior. This study aimed to investigate the relationship between mast cell density (MCD) (total, peritumoral, and intratumoral) and microvascular density (MVD), and to determine their association with clinicopathological features in 40 canine mammary carcinomas (CMCs). The mast cells (MCs) assessment was conducted by utilizing standard Giemsa staining. To evaluate angiogenesis, the immunohistochemical expression of CD31 was assessed. Results revealed that peritumoral MCD was significantly higher than intratumoral MCD (P < 0.001). Higher histological grades (Grade III) exhibited increased MVD (P = 0.09) and total/intratumoral MCD (P = 0.031 and P = 0.020, respectively). Necrosis was associated with elevated MVD (P = 0.04) and intratumoral MCD (P = 0.027). However, no correlation was found between MVD and MCD, nor were significant differences observed among histological subtypes and lymphovascular invasion. These findings indicate that mast cells might play a role in tumor progression, potentially by promoting angiogenesis. Additionally, their distribution could serve as an extra histopathological marker that, when combined with traditional grading, may enhance prognostic accuracy for CMCs. Further studies with larger cohorts are needed to highlight the complex role of mast cells in the tumor microenvironment.

Glucose-lowering treatment and antidiabetic drugs show great potential for preventing and intervening in diabetes mellitus (DM)-associated cognitive dysfunction or advanced dementia. This study delves into the role and mechanism of low-carbohydrate diet (LCD) combined with Xiaokeping (XKP) on these conditions. Type 2 diabetes (T2D) rat models were constructed via high-fat diet and streptozotocin (STZ) injection, and treated with LCD and XKP alone or in combination for 8 weeks. Body weight and indicators of blood glucose and lipids were determined. Additionally, memory and spatial learning abilities were assessed. Tissue staining of the hippocampus was used to observe pathological changes. Oxidative stress, levels of inflammatory factors, and phosphatidylinositol 3-kinase (PI3K)/Akt serine/threonine kinase (Akt) were analyzed. The results demonstrated that LCD and/or XKP treatment recovered body weight, blood glucose, and lipid balance, while enhancing spatial memory and attenuating lesion in the hippocampus of diabetic rats. LCD and/or XKP treatment increased superoxide dismutase, decreased malondialdehyde, interleukin-1β/6, and tumor necrosis factor alpha level in the hippocampus of diabetic rats, which suggests a reduction in oxidative stress and neuroinflammation. Moreover, LCD and/or XKP treatment activated the PI3K/Akt pathway. LCD combined with XKP displayed superior efficacy compared to monotherapy. Collectively, LCD combined with XKP mixture improves cognitive decline of T2D rats by suppressing oxidative stress and neuroinflammation in the hippocampus, through a mechanism involving the activation of the PI3K/Akt pathway.

Precision-cut tissue slices (PCTS), defined as thin, uniformly cut sections of fresh organs that preserve native tissue architecture and cell-cell interactions, serve as valuable ex vivo models for studying tissue physiology, toxicology, and metabolism of drug substances. However, existing PCTS preparation protocols typically focus on single-organ preparation, leaving the feasibility of sequential multi-organ slicing remains uncertain. This study evaluated the viability of rat liver and kidney slices during a 72-hour incubation period, considering factors such as sex, organ collection method, coring technique, and slicing duration. Viability was assessed based on intracellular Adenosine Triphosphate (ATP) as an indicator of metabolic activity. No sex-related differences in ATP levels were observed in 18-20-month-old rats. ATP content was also comparable between central and peripheral liver regions. In contrast, kidney slices composed of the medulla demonstrated significantly higher ATP levels than those predominantly composed of cortex, reflecting regional differences in microanatomy and metabolic adaptability. Furthermore, slices obtained from anesthetized rats showed better ATP recovery during incubation than those from euthanized rats, highlighting the importance of adequate oxygen supply maintained through blood circulation during organ collection. The sequential preparation of liver and kidney slices within 4 hours after organ collection was feasible, as slicing performed within 0-2 hours and 2-4 hours did not significantly affect viability. These findings provide practical insights for optimizing PCTS preparation supporting reliable slice viability, and enabling efficient multi-organ processing when tissue slicer capacity is limited.

Circadian rhythms are biological processes that occur in approximately 24-hour cycles in organisms. Disruption of circadian rhythms (CD) is thought to have adverse effects on many organs, including the eyes. The aim of this study was to evaluate the effects of circadian disruption on the cornea and conjunctiva, as well as the impact of two forms of omega-3 supplementation on the ocular surface, using a rat model of circadian disruption. This experimental study included 32 female Wistar albino rats, which were divided into four groups. The control group (Group 1) was maintained under normal feeding and sleeping conditions without any disruption. Circadian disruption was induced in the other three groups, which were administered saline (Group 2), fish oil (Group 3), or flaxseed oil (Group 4) via oral gavage. On the 31st day, all rats were euthanized, and corneal, conjunctival, and palpebral tissues were collected from both eyes through enucleation. Histological examination and immunohistochemical analysis of Caspase-3 (Cas-3), tumor necrosis factor-alpha (TNF-α), and PERIOD-2 (PER-2) were performed on the corneal and conjunctival tissues. Group 2 exhibited significantly thicker corneas compared to Group 1 (P < 0.001). Additionally, hyperemia, inflammatory cell infiltration in the conjunctiva, higher expression levels of Cas-3 and TNF-α, and decreased PER-2 expression were observed in the corneal and conjunctival tissues of Group 2. These pathological changes were minimal or absent in Groups 3 and 4. Notably, Group 3 showed better amelioration of these alterations compared to Group 4. Disruption of circadian rhythms can have a negative impact on the cornea and conjunctiva. Omega-3 supplementation demonstrated a significant protective effect against ocular tissue damage induced by circadian disruption. Fish oil was more effective than flaxseed oil in reducing corneal thickening, inflammation, and apoptotic marker expression, highlighting its potential as a therapeutic intervention for circadian rhythm-related ocular pathologies.

The aim of this study was to determine the effects of three linear cyanobacterial oligopeptides: microginin-FR1 (MG-FR1), anabaenopeptin-B (ANA-B), aeruginosin 98A (AER-A), aeruginosin 98B (AER-B) and one cyclic oligopeptide anabaenopeptin-B (ANA-B) on RTgill-W1 fluorescent biomarkers of cell line derived from Rainbow trout gill cells. The following cellular parameters were determined after 48-h exposure to different concentrations (205 nM, 512 nM and 1004 nM) of the oligopeptides: Hoechst and Propidium Iodide (PI) fluorescence, intracellular glutathione level and cytoskeletal F-actin structure. The study showed that AER-B and MG-FR1 were the most potent inhibitors of Hoechst fluorescence, AER-B additionally increased PI fluorescence level. MG-FR1 and ANA-B substantially reduced glutathione and F-actin fluorescence level in RTgill-W1 cells. The obtained results suggest that both linear and cyanobacterial oligopeptides should be considered as factors inducing detrimental alterations in RTgill-W1 cells, suggesting that in natural conditions exposure of fish to cyanobacterial metabolites may result in cytotoxic effects in fish gills.

Estrogen deficiency and diabetes mellitus (DM) are major endocrine and metabolic disorders contributing to musculoskeletal degeneration through oxidative stress, inflammation, and extracellular matrix remodeling. The synovial membrane, essential for joint homeostasis, is particularly vulnerable to these systemic disturbances. Autophagy, a key cellular mechanism for maintaining synovial integrity by degrading damaged organelles and proteins, may be dysregulated under such conditions; however, its regulation in response to estrogen deficiency and DM remains poorly understood. This study investigated the combined effects of estrogen deficiency and streptozotocin-induced DM on rat knee synovial tissue, focusing on histopathological changes and the immunoexpression of autophagy (Beclin-1, LC3B), angiogenic (VEGF-A), matrix remodeling (MMP-9), and inflammasome-related (NLRP3) markers. Twenty adult female Wistar rats were ovariectomized (OVX) or SHAM-operated (SHAM) and assigned to SHAM, OVX, SHAM-DM, and OVX-DM groups. DM was induced by intraperitoneal streptozotocin injection in the diabetic groups. After seven weeks, knee joints were fixed in paraformaldehyde, decalcified, and embedded in paraffin. Sections were analyzed histologically and immunohistochemically. OVX-DM rats showed pronounced synovial lining hyperplasia, fibrosis, and vascular proliferation, with increased expression of Beclin-1, LC3B, MMP-9, VEGF-A, and NLRP3. Significant positive correlations were observed among these markers and with fibrosis, vascularity, and inflammation scores. These findings suggest that estrogen deficiency and DM synergistically promote pathological synovial remodeling via activation of autophagic, angiogenic, and matrix degradation pathways.