Earticle

The use of genetically modified animals for human diseases contributes to the understanding of the pathogenesis of various diseases and aids in the search for effective treatments. Consequently, there is an emerging trend of establishing appropriate genetic animal models capable of recapitulating the crucial phenotypes of human diseases to facilitate investigations about the pathogenesis and effective treatments. Recently, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas) technology demonstrated its successful application in genetically modifying the genomes of diverse species. Furthermore, CRISPR/ Cas technology has been established as a tool for producing genetic animal models that more faithfully mimic human diseases. In this review, we have focused on the application of CRISPR/Cas technology in developing innovative genetic animal models for the study of human diseases, specifically for the investigation of disease pathogenesis and treatment. We have also discussed the current technical challenges of CRISPR technology, such as off-target effects, and reviewed the emerging strategies developed to enhance its precision. Finally, we have provided an overview of the considerable potential of advanced tools derived from the CRISPR/dead Cas9 (dCas9) system, such as base and prime editors, and explore their future applications in creating more sophisticated disease models.

Accumulating evidence indicates that the gut microbiota plays a pivotal role in overall health through complex interactions along the gut-brain axis. The present study explores the intricate relationships between the gut microbiota and the gut-brain axis, emphasizing their involvement in various neuropsychiatric disorders and the distinctive microbial compositions associated with each condition. First, the crucial role of the gut microbiota in physiological processes within the brain is discussed, with particular attention to the complex bidirectional interactions and regulatory mechanisms underlying the brain–gut–microbiome system. Second, the influence of gut dysbiosis on several neuropsychiatric disorders such as major depressive disorder (MDD), Parkinson’s disease (PD), and schizophrenia (SCZ), is extensively examined. The symptoms of each disorder are evaluated using animal behavioral assessments, which serve as the basis for analyzing the correlation between alterations in gut microbiota composition and behavioral abnormalities. Subsequently, strategies have suggested for addressing these neuropsychiatric disorders through the modulation of gut microbiota, such as probiotics and fecal microbiota transplantation (FMT), are reviewed. While existing studies provide a foundational understanding, further evidence is required to achieve a comprehensive understanding of gut microbiomebrain interactions and, consequently to develop robust and practical therapeutic and prophylactic strategies. By thoroughly reviewing the latest findings on the gut-brainaxis, this study aims to provide valuable insights for future research on the treatment and prevention of neuropsychiatric disorders based on gut microbiota.

Background: Premature ovarian insufficiency (POI) affects reproductive capacity and endocrine function in women of reproductive age. Preclinical models that reliably recapitulate POI are needed to evaluate ovarian-protective strategies. Methods: Female C57BL/6 mice received a single intraperitoneal injection of cyclophosphamide (120 mg/kg) plus busulfan (30 mg/kg). A subset received an additional cyclophosphamide booster (60 mg/kg) at week 4. Body weight, serum estradiol-2 (E2), and ovarian index (ovary weight compared to control ovary) were assessed weekly for six weeks. Follicle counts were performed on H&E-stained ovarian sections at weeks 1 through 6. Results: Animals experienced a transient ~ 5% body-weight loss in week 1 with full recovery by week 2 (p > 0.05 vs. controls). Ovarian index declined by ~ 50% in the single-injection group by week 4 and remained stable through week 6 (p < 0.05). The booster group exhibited a comparable reduction by week 4 that persisted at weeks 5 and 6 (p > 0.05), indicating maintenance, but no further exacerbation, of ovarian atrophy. Serum E2 dropped by 40% at week 6 in single-injection mice, whereas booster-injected mice showed a 74% decline by week 5 (p < 0.05). Histologically, follicle counts decreased by 84% in the single-injection group (not significant) and by 100% in the booster group by week 6 (p < 0.05). Conclusions: A single dose of cyclophosphamide and busulfan induces sustained ovarian atrophy characteristic of premature ovarian insufficiency (POI), and a delayed booster accelerates hormonal decline without further mass loss. This model offers a standardized platform for testing interventions to preserve ovarian function.

Background: Conventional two-dimensional (2D) culture systems have traditionally served as the standard platform for anticancer drug development. However, because cancer cells grow and interact within complex three-dimensional (3D) microenvironments in vivo , 2D-based assays often fail to accurately recapitulate cellular behavior and predict therapeutic efficacy. To overcome these limitations, bioengineered hydrogels have been explored to construct 3D culture systems tailored to cancer cells. Methods: As a step toward constructing a PEG-based hydrogel platform for the 3D culture of epithelial ovarian cancer, matrix metalloproteinases (MMPs) secreted by SKOV3 cells (a human ovarian cancer cell line) were analyzed at both the transcriptional and translational levels by qPCR and human MMP antibody array. Results: Among seven representative MMPs analyzed, the transcriptional expression levels of the MMP genes decreased in the following order: MMP1 > MMP2 > MMP13 > MMP10 > MMP3 > MMP8 > MMP9. However, at the protein level, only MMP1, MMP10, and MMP13 were detected, and MMP10 and MMP13 showed significantly higher secretion levels than MMP1. MMP2, MMP3, MMP8, and MMP9 were not detected at the translational levels. Conclusions: The identification of protease secreted specifically and abundantly from SKOV3 cells is important for designing MMP-cleavable crosslinkers, enabling the construction of PEG-based hydrogels responsive to cellular activity. These findings contribute to the development of a physiologically relevant 3D culture environment for ovarian cancer research and anticancer drug screening.

Background: This study evaluated the impact of intra-abdominal lidocaine and systemic meloxicam, alone and in combination, on oxidative stress biomarkers in healthy dogs undergoing elective ovariohysterectomy (OHE). Methods: Twenty-eight female dogs presenting to the Aydın Adnan Menderes University Veterinary Teaching Hospital were randomly assigned to one of four groups (n = 7 each): Control (OHE only), Lidocaine (1 mg/kg sprayed intraperitoneally at surgical sites), Meloxicam (0.2 mg/kg SC post-closure), and Lidocaine + Meloxicam (both treatments as above). Blood was sampled pre-operatively and at 2, 12, and 24 hours post-surgery. Serum concentrations of malondialdehyde (MDA), catalase (CAT), and glutathione peroxidase (GSH-Px) were quantified as indices of lipid peroxidation and antioxidant capacity. Results: In the combined-treatment group, MDA, CAT, and GSH-Px levels declined significantly over time, indicating attenuated oxidative stress. In contrast, lidocaine alone produced a significant time-dependent decrease only in CAT activity, while meloxicam alone did not significantly alter any marker. Conclusions: These findings suggest that the synergistic use of intraperitoneal lidocaine with systemic meloxicam more effectively mitigates postoperative oxidative stress than either agent alone, and may represent a beneficial analgesic–antiinflammatory strategy in canine OHE.

Background: Diazinon (DZN), a widely used organophosphorus pesticide, exerts toxic effects on the reproductive system. In this study, we investigated its effects on neonatal testicular development using mouse testicular fragments cultured in vitro. Methods: Male mouse testis fragments were cultured in vitro with varying concentrations of DZN for 5 days to assess spermatogenic toxicity. Histology, immunostaining, DHE staining, qPCR, and Western blotting were used to evaluate cellular damage, oxidative stress, and gene/protein expression. Results: Histological analysis showed that 50-100 μM diazinon (DZN) induced vacuolization of seminiferous tubules, whereas lower concentrations had no significant morphological effects. Gene and protein expression analyses revealed substantial reductions in both undifferentiated and differentiated germ cell markers (SALL4, DDX4, and SYCP3), indicating germ cell loss. Immunostaining confirmed decreased proportions of DDX4- and SYCP3-positive cells in DZN-treated testes. Additionally, DZN impaired Sertoli and Leydig cell function, as shown by downregulation of key markers (SOX9, WT1, INSL3, and 3β-HSD), verified through immunostaining and western blotting. DZN also elevated reactive oxygen species (ROS) levels, as indicated by intense DHE staining in various testicular cell types. Correspondingly, oxidative stress-related genes (NRF2, HO-1, SOD1, and catalase) and HO-1 protein levels were significantly upregulated, confirming ROS overproduction. Conclusions: Overall, these findings suggest that DZN induces germ cell loss and disrupts the Sertoli and Leydig cell functions in neonatal testes, primarily via oxidative stress and ROS-mediated damage.