Background: Prion diseases (PrDs) are fatal transmissible neurodegenerative disorders caused by misfolded prion protein, which is highly expressed in the brain. Drosophila has been employed as a model system for studying mammalian neurodegenerative diseases.

Methods: Drosophila transgenic for hamster prion protein (HaPrP) was generated by Valium20 transformation. Locomotion, longevity, protease resistance, and histology were assessed, and nontargeted metabolomics analyses were performed to investigate the changes in Drosophila metabolism with the HaPrP expression and metformin treatment.

Results: The Drosophila model exhibited pan-neuronal expression of HaPrP, with expression levels increasing with age. Flies displayed reduced climbing ability, shortened lifespan, and vacuolar structures in the brain. Additionally, HaPrP expressed in older flies demonstrated resistance to digestion by 5 μg/mL Proteinase K. The Drosophila model also displayed alterations in protein, lipid, and carbohydrate metabolism. We hypothesize that glutamate, N-acetylaspartate, ceramide, phosphatidylethanolamine, dihydroxyacetone phosphate, ribose-5-phosphate, and pyruvate are key metabolites potentially related to PrDs. Metformin improved locomotor activity, reduced PrP^res formation, and ameliorated mitochondrial dysfunction in flies, which may be associated with alterations in succinate, pyruvate, choline, and sphingomyelin levels.

Conclusions: We generated a Drosophila model of PrDs that recapitulates key pathological features observed in mammals. Preliminary applications have demonstrated that the Drosophila model is suitable for PrDs research and the high-throughput screening of potential therapeutic compounds.

Background: Qi pi pill (QPP), which contains Renshen, Baizhu, Fuling, Gancao, Chenpi, Shanyao, Lianzi, Shanzha, Liushenqu, Maiya, and Zexie, was recommended for preventing and treating COVID-19 in Shandong Province (China). However, the mechanism by which QPP treats infectious diseases remains unclear. This study aims to investigate the therapeutic effect of QPP in vitro and on acute influenza infection in mice, exploring its mechanism of action against influenza A virus (IAV).

Methods: The in vitro activity of QPP was assessed using dose–response curve analysis and titer reduction assay, and its antiviral mechanism was identified in vitro by real-time polymerase chain reaction (PCR), time-of-addition, and enzymatic assays. The antiviral efficacy of QPP was further evaluated in vivo using BALB/c mice infected with IAV. At the same time, each single Chinese herbal medicine in QPP was evaluated to preliminarily identify those with antiviral effects.

Results: In vitro results showed that QPP exhibited a higher potency antiviral effect against both influenza A and B viruses, inhibiting viral RNA replication and release by targeting RNA-dependent RNA polymerase and neuraminidase. Additionally, QPP significantly decreased the expression of inflammatory cytokines in A549 cells. In vivo study revealed that QPP significantly reduced the lung index and viral load in lung tissue of mice infected with IAV. Renshen, Gancao, Zexie, and Lianzi were the Chinese herbal medicines from QPP that showed anti-IAV activity.

Conclusion: The antiviral activity of QPP targets IAV replication and release, cytokine modulation in host cells, and provides protection in mice with acute influenza infection.

Background: Vaccinia virus (VACV) and mpox virus (MPXV) belong to the orthopoxvirus genus and share high genetic similarity, making VACV widely used in the mpox pandemic. CAST/EiJ mice have been widely used for studying orthopoxvirus infection. However, the histopathological features of CAST/EiJ mice with mpox virus (MPXV) and vaccinia virus (VACV) infections have not been fully elucidated.

Methods: Four group of CAST/EiJ mice were challenged with low-dose VACV (10³ PFU, VACV-L), high-dose VACV (10⁶ PFU, VACV-H), MPXV (10⁶ PFU) or PBS via intraperitoneal route, and the disease signs and body weight were monitored daily. Subsequently, viral loads and titers in the blood and spleen of CAST/EiJ mice were analyzed via qPCR and TCID₅₀ assay. Finally, the spleen samples were analyzed for histopathological, immunohistochemical and RNA-seq.

Results: Herein, we found that VACV-L and MPXV caused splenomegaly via the intraperitoneal route, whereas VACV-H caused rapid lethality with limited splenomegaly. Transcriptome analysis from spleen revealed significant differences in gene expression between VACV-L and VACV-H groups, but the differentially expressed genes induced by splenomegaly between VACV-L and MPXV groups were highly similar. Furthermore, pathway enrichment analysis demonstrated that the VACV-L, VACV-H, and MPXV groups were all associated with the calcium, MAPK, and PI3K-Akt signaling pathway. Compared to the lethal infection observed in VACV-H group, the splenomegaly in the VACV-L and MPXV groups was characterized by extramedullary hematopoiesis and increased macrophages infiltration in the red pulp. Transcriptome analysis of the spleen demonstrated that the Wnt, tumor necrosis factor (TNF), and transforming growth factor β (TGF-β) signaling pathways may promote splenomegaly by modulating granulocyte infiltration and inflammatory responses. Compared to VACV-L group, the limited splenomegaly but lethality in VACV-H-infected mice might be associated with extensive splenic necrosis, diffuse congestion, and hemorrhage in the red pulp, as well as changes in the cGMP-PKG, Ras signaling, and Fc gamma R-mediated phagocytosis pathways.

Conclusions: Our findings systematically compared the pathogenicity of VACV and MPXV in CAST/EiJ mice, incorporating splenic transcriptome analysis to provide insights into the potential molecular mechanism behind orthopoxvirus-induced splenomegaly in CAST/EiJ mice.

Post-stroke depression (PSD) is a common psychiatric complication affecting nearly one-third of stroke survivors, leading to increased disability, mortality, and cognitive decline. Traditional Chinese Medicine (TCM) has proven effective in treating PSD through syndrome differentiation, yet existing animal models primarily reflect Western medical concepts and fail to incorporate the TCM principle of “同病异治” (treating the same disease with different methods). This paper provides a review of the current methods for constructing animal models of post-stroke depression (PSD) from the perspective of Traditional Chinese Medicine (TCM) syndrome differentiation and proposes multi-dimensional assessment indicators. By integrating TCM theories with modern biomedical techniques, this study offers a comprehensive framework for deepening the understanding of the pathogenesis and therapeutic evaluation of PSD. This approach not only contributes to advancing PSD research but also paves the way for innovative treatment strategies that combine traditional and modern medicine.

Background: SOX6 has been shown to play a crucial role in the development of the cardiovascular system. However, its potential role in hypertension and vascular function remains unclear.

Methods: In vascular smooth muscle cells (VSMCs), we employed gain- and loss-of-function approaches combined with RNA sequencing, autophagy flux assessment, and phenotype characterization. Additionally, we established a mouse model with Sox6 overexpression via adeno-associated virus 2 (AAV2) to validate the findings in vivo.

Results: We validated the increased expression of SOX6 in hypertension both in vitro and in vivo. Genetic silencing of Sox6 in VSMCs attenuated the phenotypic switching induced by angiotensin II. Conversely, in vivo overexpression of Sox6 led to a significant elevation in blood pressure and promoted vascular remodeling. Mechanistically, SOX6 was shown to regulate phenotypic switching via an autophagy-dependent pathway. Specifically, Sox6 overexpression augmented VSMC autophagy and facilitated phenotypic switching, whereas Sox6 knockdown yielded opposite outcomes. Modulation of autophagy using 3-MA or RAPA could effectively counteract the effect mediated by SOX6.

Conclusions: Our findings revealed that SOX6 regulates VSMC plasticity and elevates blood pressure by activating autophagy. Therefore, SOX6 inhibition potentially represents a novel strategy for treating hypertension and vascular remodeling.

Background: Chaetomorpha aerea, a marine green alga, has drawn attention because of its rich phytochemical constituents and therapeutic benefits. Using an integrated approach that combined in vitro, in vivo, and in silico approaches, this work examined the antioxidant, anti-inflammatory, and antidiabetic qualities of acetone extract of C. aerea (AECA).

Methods: Total phenolic and flavonoid concentrations of AECA were measured. Antioxidant activity was assessed using the DPPH and ABTS free radical scavenging assays. In vitro protein denaturation and in vivo carrageenan-induced paw edema models were employed to evaluate the anti-inflammatory potential, whereas antidiabetic activity was assessed using in vitro α-amylase inhibition and in vivo oral glucose tolerance test (OGTT). Molecular docking and ADME/T analysis were employed to further analyze bioactive compounds identified using gas chromatography–mass spectrometry (GC–MS).

Result: Antioxidant activity demonstrated a minimum inhibitory concentration (IC₅₀) of 107.44 μg/mL for DPPH and 118.23 μg/mL for ABTS. In vitro anti-inflammatory assays indicated a suppression of protein denaturation at a concentration of 102 μg/mL (IC₅₀), where AECA (400 mg/kg) resulted in a 27% reduction in paw edema at 6 h in the mouse model. In vitro antidiabetic test indicated α-amylase inhibition with an IC₅₀ value of 70.72 μg/mL, and in the OGTT, a significant lowering of blood glucose was recorded at 120 min in mice. Strong binding affinities were observed for stigmasta-5,24(28)-dien-3-ol, identified using GC–MS, with values of −9.9 kcal/mol for α-amylase and − 8.0 kcal/mol for cyclooxygenase-2.

Conclusion: C. aerea serves as an effective natural remedy for oxidative stress, inflammation, and hyperglycemia. These findings advocate for further clinical and mechanistic investigations to optimize therapeutic efficacy.

Background: Multiple sclerosis (MS) is a chronic disease of the central nervous system (CNS), exhibiting hallmarks of both inflammation and neurodegeneration and with limited treatment options. The intricate nature of MS pathophysiology and its variable progression pose severe challenges for the development of effective therapies. The experimental autoimmune encephalomyelitis (EAE) MS model, in its most common form, is an aggressive disease, which is not representative of the MS course and offers a limited time window for drug evaluation. This study aimed to generate an attenuated EAE variant, which extends the clinical testing window while preserving the high incidence of the standard EAE model.

Methods: Components of the EAE induction protocol were titrated to develop a milder disease profile. In a subsequent drug trial using the MS medication fingolimod hydrochloride (FTY, Gilenya), the new variant was validated under prophylactic and therapeutic treatment regimens.

Results: The attenuated EAE variant retains the standard hallmarks of neuroinflammation and, crucially, significantly extends the time frame for clinical drug testing. Unlike the standard variant, where FTY efficacy could only be demonstrated by prophylactic treatment, the attenuated variant facilitated differentiation of drug effects by therapeutic treatment initiated early in the acute phase of disease.

Conclusion: The new EAE variant is suitable for use in preclinical assessment of candidate therapeutics and the identification of targetable molecular mechanisms underpinning disease development and progression. This study illustrates the importance of optimizing and refining the experimental tool to enhance the translational success of the candidate therapeutics for MS.

Background: This study aimed to discover whether Cistanche tubulosa affects the AKT/CASP3 pathway by regulating m6A methylation, to exert a protective effect against peripheral nerve injury in a Parkinson's Disease (PD) mouse model.

Methods: In this study, network pharmacology analysis and the molecular docking virtual screening technique was used to filter Acteoside (Act), a potential neuroprotective agent of active components in Cistanche tubulosa. A PD-related peripheral neuropathy mouse model was established by MPTP induction, followed by 21 days treatment of oral Act (25, 50, and 100 mg kg⁻¹). Pole climbing, automatic avoidance ability and hot plate sensory tests were evaluated to determine behavioral changes caused by central and peripheral nerve injury. The pathological alterations of dorsal root ganglion tissue and the protein levels of IL-6, AKT, and CASP3 under Act intervention, as well as the dynamic changes of FTO, METTL3, and YTHDF2 which are closely related to m6A methylation, were comprehensively analyzed to observe the peripheral nerve protective efficacy of Act.

Results: The results showed that peripheral neuropathy occurring with PD in the mouse model, which could be verified by behavioral tests and pathological histological changes. In addition to the previously established protective effect of Act on dopaminergic neurons in substantia nigra (SN), extensive follow-up studies demonstrated that Act effectively induced m6A methylation, which could further regulate the AKT/CASP3 pathway to play a therapeutic role. In this study, medium and high doses of Act played more obvious therapeutic roles.

Conclusion: These findings suggest that Act may regulate the severity of peripheral nerve injury under the activation of the AKT/CASP3 signaling pathway by balancing the methylation level of m6A. These results provide a theoretical basis and experimental evidence for further research on the protective effect of Cistanche tubulosa on both the central and peripheral nerves in the treatment of PD.

Background: The two-kidney two-clip (2K2C) rat model is widely used in hypertension, stroke, and drug studies. However, knowledge of this model is quite limited.

Methods: In this study, U-shaped silver clips with inner diameters of 0.3, 0.25, and 0.2 mm were used to narrow bilateral renal arteries, inducing different extents of blood flow reduction for preparing 2K2C rats. Blood pressure (BP) was continuously measured during 2K2C procedures until 10 h after 2K2C, and long-term assessments of BP, organ damage, and serum biochemistry were performed at 1, 2, 4, and 6 weeks.

Results: BP increased immediately when the 2K2C rats regained consciousness from anesthesia, with a 100% incidence of hypertension on the day of operation, and increased to a plateau at 2 weeks for 0.2 mm clips (248 mmHg), 4 weeks for 0.25 mm clips (219 mmHg), and perhaps 6 weeks for 0.3 mm clips (206 mmHg). BP levels displayed a negative correlation with clip sizes. Organ damage, including aortic and cardiac hypertrophy, cerebrovascular and cardiovascular injury, and renal atrophy or compensatory enlargement, occurred as early as 1 or 2 weeks after 2K2C. Routine serum biochemistry indicated organ dysfunction primarily at 4–6 weeks in 0.2 mm clips, whereas aspartate aminotransferase appeared a sensitive biomarker for early organ damage.

Conclusion: 2K2C can produce severe hypertension and multi-organ damage in a short time. 2K2C rats in 0.2 mm clips can be used as a hypertensive emergency model. This study provides crucial insights for guiding the development of targeted interventions.

Background: The Cre/loxP system is most popular in mice, but its application in rats has largely lagged far behind. The rat is vital laboratory animal, especially in toxicological and neurological studies. Generating genetic tools to manipulate neurons in rats could benefit neurological research.

Methods: Using the CRISPR/Cas9 system, we inserted a Cre cassette into endogenous Thy1 and NeuN loci. Thy1-Cre rats featured a downstream P2A-linked insertion, while NeuN-Cre was inserted at the transcriptional start site. The Cre activity was assessed by crossing with a Cre reporter (Rosa26^imCherry) rat and through analyzing mCherry expression patterns. The specificity of cell type was further confirmed by immunofluorescence with NeuN antibody. Phenotypic consequences were assessed by crossing with ND1^LSL rats to deplete ND1, followed by monitoring weight/survival and conducting motor function tests.

Results: We generated two neuron-specific rats (Thy1-Cre and NeuN-Cre), which exhibited high neuron-specific Cre expression in brain and spinal cord with minor leakage in other tissues. Thy1-Cre showed minor leakage in spleen, lung and kidney while NeuN-Cre showed minor leakage in spleen and kidney. ND1^Thy1-Cre and ND1^NeuN-Cre rats both showed decreased body weights and survival times. The ND1^NeuN-Cre rats died within two weeks, while ND1^Thy1-Cre rats lived longer with impaired motor function.

Conclusions: We successfully generated two neuron-specific NeuN-Cre and Thy1-Cre rats, and systemically analyzed their expression pattern.

Background: Hemorrhagic expansion into the fourth ventricle is an independent risk factor for poor outcomes in intraventricular hemorrhage (IVH) patients. However, to date, available animal models of IVH are limited to models of supratentorial ventricular hemorrhage, and there are no specific models of fourth ventricle hemorrhage. This limitation hinders comprehensive basic research and the understanding of the pathophysiological changes that occur following fourth ventricle hemorrhage. Therefore, the development of an animal model of fourth ventricle hemorrhage is highly important.

Methods: In this study, a novel rat model of fourth ventricle hemorrhage was established via autologous blood injection through the foramen of Magendie. Anesthetized rats were positioned in a stereotaxic apparatus with their heads tilted downward at an angle of approximately 20° relative to the vertical axis. A needle was inserted through the foramen, and autologous blood obtained from the rat's heart was injected into the fourth ventricle via a microinfusion pump. Systematic evaluations of the model were conducted using small-animal magnetic resonance imaging, histopathological analysis, and neurological function assessment.

Results: The rats developed stable and reproducible fourth ventricle hematomas and ventricular dilation. They also exhibited acute-phase hydrocephalus and pathological features of perilesional brain tissue injury, with observed neurological deficits comparable to patients with fourth ventricle hemorrhage.

Conclusion: This model successfully recapitulates the clinicopathological and pathophysiological characteristics of patients with fourth ventricle hemorrhage and can be utilized for further investigation into the pathophysiological mechanisms underlying posthemorrhagic hydrocephalus and perilesional brainstem tissue injury.

Background: During the establishment of a model of acute kidney injury (AKI) in pigs, we observed a high prevalence of malignant hyperthermia (MH). These complications led us to refine the anesthetic protocol. This publication describes the impact of the choice of anesthetics on the results obtained.

Methods: Pigs were euthanized at the end of the procedure, without recovery from anesthesia. Three anesthetic protocols were used: sevoflurane inhalation (ProtocolA, n = 5), a combination of ketamine, medetomidine and diazepam by intravenous infusion (ProtocolB, n = 5), and a combination of ketamine, diazepam, medetomidine, glucose, and noradrenaline (ProtocolC, n = 5). All pigs received morphine for analgesia. AKI was induced by interrupting renal perfusion for 90 min. MH was diagnosed based on clinical and biological parameters.

Results: All MH pigs belonged to ProtocolA. MH pigs showed significantly higher maximum rectal temperature (p = 0.04), maximum expired carbon dioxide (CO₂; p = 0.04), maximum heart rate (HR; p = 0.03), plasma concentration of creatinine and potassium (p < 0.0001). Protocol A pigs had a significantly higher maximum HR (p = 0.01) and hyperkalemia compared to the two other groups (ProtocolB, p = 0.005 and ProtocolC, p < 0.0001). Pigs from ProtocolA had a significantly lower minimum mean arterial pressure (MAP) than ProtocolC group (p = 0.03) and MAP remained below 60 mmHg for longer (p = 0.004). In ProtocolB, minimum glycemia was lower than other groups (p = 0.01).

Conclusion: Sevoflurane use was associated with the occurrence of MH, hemodynamic alterations and changes in plasma concentration of creatinine and potassium. These modifications can have a major impact on the validation of an experimental AKI model.

Background: The efficacy of balloon angioplasty for treating peripheral artery disease is influenced by various factors, some of them not yet totally understood. This study aimed to evaluate the role of elastin content in vascular responses 28 days post-angioplasty using uncoated and paclitaxel-coated balloons with the same platform in femoral arteries of a healthy porcine model.

Methods: Eight animals underwent balloon angioplasty on the external and internal branches of femoral arteries. Histopathologic evaluation was conducted at follow-up to assess the elastin content, vascular damage, morphological features, and neointimal formation.

Results: The elastin content was significantly higher in the external than in the internal femoral artery (p = 0.0014). After balloon angioplasty, it was inversely correlated with vascular injury score (ρ = −0.4510, p = 0.0096), neointimal inflammation (ρ = −0.3352, p = 0.0607), transmural (ρ = −0.4474, p = 0.0103) and circumferential (ρ = −0.4591, p = 0.0082) smooth muscle cell loss, presence of proteoglycans (ρ = −0.5172, p = 0.0024), fibrin deposition (ρ = −0.3496, p = 0.0499), and adventitial fibrosis (ρ = −0.6229, p = 0.0002). Neointimal formation inhibition with paclitaxel was evident only in arteries with disruption of the internal elastic lamina, with a significant smaller neointimal area in arteries treated with paclitaxel-coated balloons compared to uncoated balloons (median [Q1–Q3]: 10.25 [7.49–15.64] vs. 24.44 [18.96–30.52], p = 0.0434).

Conclusions: Elastin content varies between branches of the femoral artery and significantly influences the integrity of the internal elastic lamina, the vessel's adaptive response, and paclitaxel efficacy after balloon angioplasty.

Oral squamous cell carcinoma (OSCC) constitutes 90% of oral tumors. Advanced cases severely impair patients' life quality of life due to anatomical location and limited therapies. Conventional treatments often induce drug resistance or recurrence. Patient-derived xenograft (PDX) models are widely used to simulate tumor progression and drug responses, serving as translational tools for precision medicine. This study aimed to establish drug-resistant OSCC PDX models. Human OSCC tissues were transplanted into immunodeficient mice and passaged (P1–P2). At P2 (tumor volume: 40–80 mm³), mice received cisplatin (1 mg/kg, three times/week) with cetuximab (1 mg/kg, weekly), GSK690693 (10 mg/kg, five times/week), or rapamycin (4 mg/kg, five times/week). PDX tissues from groups with less-therapeutic response (manifested as larger tumor volumes) were serially passaged to assess treatment efficacy. Tumor tissues with diminished drug sensitivity underwent histopathological analysis and identified stability of their tumor characteristics using hematoxylin–eosin (HE) and immunohistochemical staining after one additional passage and retreatment. Results demonstrated that successive passaging accelerates tumor growth. First-generation treatments showed universal sensitivity. At P2, cisplatin–cetuximab and rapamycin groups remained sensitive, whereas GSK690693 efficacy declined. Continued passaging of GSK690693-treated tumors confirmed resistance, as evidenced by exhibiting enhanced malignant characteristics at histological level. The GSK690693-resistant model was established first, whereas resistant models of other treatment groups were established according to similar protocols. These findings suggest that sequential passaging and drug exposure in PDX models recapitulated clinical tumor evolution, enabling the development of drug-resistant OSCC models. This study can offer methodological insights for precision therapy of OSCC.

This study aims to conduct a comparative study of two strains of laboratory rats, hooded and Wistar, to select a suitable alternative to nude rats for ovarian tissue xenotransplantation. The study investigated the effects of ovarian tissue transplantation using three experimental groups: (1) the control group receiving vitrified-warmed human ovarian tissue, (2) the OTW (ovarian tissue transplantation in Wistar) group with human ovarian tissues in Wistar rats, and (3) the OTH (ovarian tissue transplantation in hooded) group with ovarian tissues transplanted into hooded rats. A total of 12 rats (6 each from the two transplantation groups) were used, with tissue samples implanted in the dorsal neck muscles. Outcomes were evaluated using histological analyses, including hematoxylin and eosin and Masson's trichrome staining, as well as immunohistochemical assessments of CD31 for angiogenesis, fibrosis, and necrosis. The OTH group exhibited more blood vessel numbers and the Vegf gene expression than both the OTW and control groups, with significantly lower levels of fibrosis and necrosis compared to the OTW and control groups. The study indicates that hooded rats could serve as a valuable alternative for specific research when nude mice are not available. Their immune systems are more compatible with human ovarian tissue transplants than Wistar rats, and they exhibit calmer behaviors, making them better suited for laboratory work.

Although Steen's solution plays a crucial role in maintaining vascular oncotic pressure and reducing pulmonary edema due to its high concentration of human serum albumin during ex vivo lung perfusion (EVLP), it incurs high costs and potential immune responses in rat models. In our preliminary research, donor lungs perfused with Steen's solution exhibited significant lung injury post transplantation, likely due to a strong immune response elicited by the high concentration of xenogenic proteins upon reperfusion. This study explores a low-cost and efficient perfusate composition for use in rat EVLP and lung transplant models. The modified perfusate employed in this study uses artificial colloids as the primary component, with an albumin concentration of only 2%, demonstrating comparable physiological parameters, metabolic levels, and pathological changes to those of Steen's solution during EVLP. Moreover, the modified composition exhibited good biocompatibility in transplantation, with minimal lung injury. In summary, the proposed perfusate offers a safer and more economical alternative for establishing rat EVLP and lung transplant models, facilitating the advancement of related research.