Background: The maturation of the immune system is critical during early life, as it involves the differentiation, maturation, and establishment of immune tolerance of immune cells. This process is influenced not only by genetic factors but also by environmental factors, particularly the symbiotic microbiota. Bifidobacterium animalis subsp. lactis (BB-12), originally found in dairy products, is widely used in infant formula and dietary supplements. However, its role and mechanisms in immune development during early life remain unclear.
Methods: Using GF mice as the experimental model, B. animalis subsp. lactis BB-12 was administered via gavage during early life. In the juvenile stage, changes in T-cell subsets in the spleen, thymus, and gut intraepithelial lymphocytes (IEL) were assessed using spectral flow cytometry. Additionally, targeted metabolomics analysis of tryptophan metabolism and short-chain fatty acid pathways in colonic tissue was conducted to explore how B. animalis subsp. lactis BB-12 influences the immune system through gut microbiota metabolism.
Results: BB-12 effectively modulates the gut immune microenvironment, leading to beneficial changes in T-cell subsets in key immune tissues such as the spleen, thymus, and gut IELs. Metabolomics analysis further supports these findings by showing that BB-12 intervention greatly increased the production of tryptophan derivatives and acetic acid in the colon of GF mice.
Conclusion: The findings provide theoretical evidence for the role of B. animalis subsp. lactis in immune system development and support its application in dietary supplements, suggesting potential as a component for infant immune health and in preventing immune-related diseases.
Background: The composition of the intestinal flora and the resulting metabolites affect patients' sleep after surgery.
Methods: We intended to elucidate the mechanisms by which disordered intestinal flora modulate the pathophysiology of postoperative sleep disturbances in hosts. In this study, we explored the impacts of anesthesia, surgery, and postoperative sleep duration on the fecal microbiota and metabolites of individuals classified postprocedurally as poor sleepers (PS) and good sleepers (GS), as diagnosed by the bispectral index. We also performed fecal microbiota transplantation in pseudo-germ-free (PGF) rats and applied Western blotting, immunohistochemistry, and gut permeability analyses to identify the potential mechanism of its effect.
Results: Research finding shows the PS group had significantly higher postoperative stool levels of the metabolites tryptophan and kynurenine than the GS group. PGF rats that received gut microbiota from PSs exhibited less rapid eye movement (REM) sleep than those that received GS microbiota (GS-PGF: 11.4% ± 1.6%, PS-PGF: 4.8% ± 2.0%, p < 0.001). Measurement of 5-hydroxytryptophan (5-HTP) levels in the stool, serum, and prefrontal cortex (PFC) indicated that altered 5-HTP levels, including reduced levels in the PFC, caused sleep loss in PGF rats transplanted with PS gut flora. Through the brain–gut axis, the inactivity of tryptophan hydroxylase 1 (TPH1) and TPH2 in the colon and PFC, respectively, caused a loss of REM sleep in PGF rats and decreased the 5-HTP level in the PFC.
Conclusions: These findings indicate that postoperative gut dysbiosis and defective 5-HTP metabolism may cause postoperative sleep disturbances. Clinicians and sleep researchers may gain new insights from this study.
Background: Qiwenghuangbo powder (QP), composed of Astragalus, Phellodendron chinensis, and Radix pulsatilla, is a traditional Chinese herbal formula, but its effects on weaned piglets remained unclear.
Methods: Weaned piglets fed with 0.5 kg/t QP (QP1), 1 kg/t QP (QP2), low-zinc oxide (ZnO; negative control), and high-ZnO (positive control) diets in two phases, respectively, and the growth performance, intestinal morphology, cytokines, and microbial communities were profiled. The mouse models of colitis induced by Citrobacter rodentium and dextran sulfate sodium (DSS) were employed to elucidate the potential role of QP-fed enriched key species.
Results: Dietary 1.0 kg/t QP alleviated diarrhea and inflammation and improved intestinal development and growth performance of weaned piglets. Moreover, this dietary intervention notably altered microbiota composition, characterized by the enrichment of Limosilactobacillus reuteri. Furthermore, out of three isolated L. reuteri, two strains could alleviate pathogen infection and intestinal inflammation, respectively. Specifically, the anti-inflammatory effect of one strain was achieved by promoting the colonization resistance of C. rodentium as significantly reduced pathogen loads. The other strain mitigated DSS-induced colitis by enhancing the goblet cell function and inhibiting the secretion of pro-inflammatory cytokines, particularly interleukin-1β (IL-1ß) and tumor necrosis factor-α (TNF-α).
Conclusions: Dietary QP improved the growth performance and intestinal health of weaned piglets by promoting the colonization of L. reuteri. The isolated commensal L. reuteri control colitis in a strain-specific mechanism, highlighting the potential of QP and L. reuteri in providing evidence for gut health promotion.
Cell lineage tracing is a key technology for describing the developmental history of individual progenitor cells and assembling them to form a lineage development tree. However, traditional methods have limitations of poor stability and insufficient resolution. As an efficient and flexible gene editing tool, CRISPR-Cas9 system has been widely used in biological research. Furthermore, CRISPR-Cas9 gene editing-based tracing methods can introduce fluorescent proteins, reporter genes, or DNA barcodes for high-throughput sequencing, enabling precise lineage analysis, significantly improving precision and resolution, and expanding its application range. In this review, we summarize applications of CRISPR-Cas9 system in cell lineage tracing, with special emphasis on its successful applications in traditional model animals (e.g., zebrafish and mice), large animal models (pigs), and human cells or organoids. We also discussed its potential prospects and challenges in xenotransplantation and regenerative medicine.
Therapeutic antibodies are valued for their high specificity and selectivity in immunotherapy. However, the potential toxicity they may elicit underscores the necessity of assessing their preclinical efficacy and safety using suitable animal models. In this context, we review the various categories and applications of humanized mice, which have been engrafted with human cells or tissues to mimic the human immune system. These models are extensively utilized in the nonclinical assessment and development of various antibody drugs, acting as a conduit to clinical research. However, several challenges remain, including the limited lifespan of humanized mice, inadequate engraftment of human cells, and the rudimentary nature of the immune environment in these models. The development of humanized immune system models in mice presents both opportunities and challenges, potentially leading to new insights into the evolution and application of antibody therapeutics.
Background: Subcortical ischemic vascular dementia (SIVD) is a common subtype of vascular dementia. Currently, the bilateral common carotid artery stenosis (BCAS) mouse model is the most suitable SIVD rodent model. In this study, we investigated the functional and structural impairments in the hippocampus 1 month after BCAS.
Methods: We used behavioral tests, laser speckle flowmetry, long-term potentiation, histochemical staining, molecular experiments, and voxel-based morphometry to evaluate the hippocampal impairments.
Results: Behavioral studies revealed that BCAS mice exhibited worse performance. Laser speckle flowmetry detected an obvious decrease in cerebral blood flow. The synaptic plasticity of the perforant path-dentate gyrus pathway was inhibited. Decreased fractional anisotropy and increased mean diffusivity were detected in the hippocampus via diffusion tensor imaging data. A reduction in gray matter volume, which was most prominent in the hippocampus and its surrounding areas, was detected via voxel-based morphometry analysis. Impairments in cell morphology and myelin integrity were validated using histochemical staining and molecular biology techniques. In addition, the numbers of GFAP+ astrocytes and Iba1+ microglia increased in the hippocampus.
Conclusions: Overall, our study demonstrates early functional and structural impairments in the hippocampus contributing to learning and memory deficits after 1 month of BCAS, indicating that the hippocampus is vulnerable to chronic cerebral ischemia.
Background: Cannabidiol (CBD) has numerous therapeutic properties, and is used to treat neurological conditions, such as neuroinflammation. However, the optimal dose of CBD to penetrate the brain requires further investigation. The primary aim of this study was to use a mouse model and the intrabuccal route for CBD administration to determine the optimal dose at which CBD can penetrate the brain. The secondary aim was to determine whether sex is a confounding factor.
Methods: Thirty adult Kramnik mice, divided equally into three groups, were administered CBD oil intrabuccally at three doses—10, 20, and 30 mg/kg, euthanized 6 h later, and whole brain, urine, and blood samples were collected. Liquid chromatography with tandem mass spectrometry was used to analyze the collected samples.
Results: CBD and its three metabolites—7-carboxy cannabidiol (7-COOH-CBD), 7-hydroxy cannabidiol (7-OH-CBD) and 6-hydroxy cannabidiol (6-OH-CBD), were identified and quantified in all samples. The 10 and 20 mg/kg doses of CBD produced similar results in the brain, but the group given the 10 mg/kg dose had the least variation. The 30 mg/kg dose yielded the highest abundance of CBD and its metabolites in all samples, but also the greatest variation. Sex only became a confounding factor at 30 mg/kg.
Conclusions: This study shows that the intrabuccal route of CBD administration is reliable and the 10 mg/kg dose of CBD is recommended in mice because there were good CBD metabolite concentrations in all samples, with the least variation among the doses, and sex was not a confounder at 10 mg/kg.
Background: Dry eye disease (DED) predominantly results from elevated tear film osmolarity, which can not only cause ocular inconvenience but may lead to visual impairments, severely compromising patient well-being and exerting substantial economic burdens as well. Astaxanthin (AST), a member of the xanthophylls and recognized for its robust abilities to combat inflammation and oxidation, is a common dietary supplement. Nonetheless, the precise molecular pathways through which AST influences DED are still poorly understood.
Methods: Therapeutic targets for AST were identified using data from the GeneCards, PharmMapper, and Swiss Target Prediction databases, and STITCH datasets. Similarly, targets for dry eye disease (DED) were delineated leveraging resources such as the Therapeutic Target Database (TTD), DisGeNET, GeneCards, and OMIM databases, and DrugBank datasets. Interactions among shared targets were charted and displayed using CytoScape 3.9.0. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses were conducted to elucidate the functions of pivotal targets within the protein–protein interaction network. Molecular interactions between AST and key targets were confirmed through molecular docking using AutoDock and PyMOL. Molecular dynamics simulations were performed using GROMACS 2022.3. Viability of human corneal epithelial cells (hCEC) was assessed across varying concentrations of AST. A mouse model of experimental DED was developed using 0.1% benzalkonium chloride (BAC), and the animals were administered 100 mg/kg/day of AST orally for 7 days. The efficacy of the treatments was assessed through a series of diagnostic tests to evaluate the condition of the ocular surface after the interventions. The levels of inflammation and oxidative stress were quantitatively assessed using methods such as reverse transcription-polymerase chain reaction (RT-PCR), Western blot, and immunofluorescence staining.
Results: Network pharmacology suggests that AST may alleviate DED by influencing oxidation–reduction signaling pathways and reducing oxidative stress provoked by BAC. In vivo experiments demonstrated an improved overall condition in AST-administered mice in contrast to the control group. Immunofluorescence staining analyses indicated a decrease in Keap1 protein in the corneal tissues of AST-treated mice and a significant increase in Nrf2 and HO-1 protein. In vitro studies demonstrated that AST significantly enhanced cell viability and suppressed reactive oxygen species expression under hyperosmotic (HS) conditions, thereby protecting the human corneal epithelium.
Conclusion: AST is capable of shielding mice from BAC-induced DED, decelerating the progression of DED, and mitigating oxidative stress damage under HS conditions in hCEC cells. The protective impact of AST on DED may operate through stimulating the Keap1-Nrf2/HO-1 signaling pathway. Our research findings indicate that AST may be a promising treatment for DED, offering new insights into DED treatment.
Background: Noninfectious uveitis, a chronic ocular inflammatory disease, is characterized by the activation of immune cells in the eye, with most studies focusing on the role of the adaptive immune system in the disease. However, limited data exist on the potential contribution of the innate immune system, specifically the nucleotide-binding oligomerization domain and leucine-rich repeat receptor-3 (NLRP3) inflammasome pathway. This pathway has previously been identified as a driver of inflammation in several low-grade, progressive inflammatory eye diseases such as diabetic retinopathy. The aim of this study was to determine whether the NLRP3 inflammasome pathway plays a role in the pathogenesis and chronicity of experimental autoimmune uveitis (EAU).
Methods: EAU was induced in C57BL/6J mice via intraperitoneal pertussis toxin and subcutaneous interphotoreceptor retinoid-binding protein injections. After 12 weeks, eyes were enucleated, and whole eye sections were assessed for inflammasome, macrophage, and microglial markers in the retina, ciliary body, and cornea using immunohistochemistry.
Results: Our study confirmed higher NLRP3 inflammasome activation (increased expression of NLRP3 and cleaved caspase 1 labeling) in EAU mouse retinas compared to controls. This correlated with increased astrogliosis and microglial activation throughout the eye. Migratory innate and adaptive peripheral immune cells (macrophages and leukocytes) were also found within the retina and ciliary body of EAU mice. Connexin43 proteins, which form hexameric hemichannels that can release adenosine triphosphate (ATP), an upstream inflammasome priming signal, were also found upregulated in the retina and cornea of EAU mice.
Conclusion: Overall, our findings support the idea that in the EAU model there is active inflammation, even 12 weeks post induction, and that it can be correlated to inflammasome activation. This contributes to the pathogenesis and chronicity of noninfectious uveitis, and our results emphasize that targeting the inflammasome pathway could be efficacious for noninfectious uveitis treatment.
Background: Fuke Huahuang formulation (FHF) is widely used in the treatment of vaginitis, with clinical evidence indicating its promising anti-inflammatory properties.
Methods: We explored the bioactive components and potential mechanisms of FHF for treating vaginitis, and reveal its pharmacological activities against vaginitis.
Results: A total of 12 anti-inflammatory components in FHF and 584 pharmacological targets were identified. Furthermore, 1427 vaginitis-associated targets were identified, and 184 intersection targets between FHF and vaginitis were constructed for network analysis. Gene Ontology and pathway analysis revealed that the therapeutical targets of FHF against vaginitis are involved in modulating inflammatory stress, enhancing immunoregulation, reconstructing the microenvironment, and suppressing cell damage. Molecular docking analysis further suggested the possible direct binding of the bioactive compounds of FHF (fumarine) to the core targets, including AKT Serine/Threonine Kinase 1 (AKT1), Signal Transducer and Activator of Transcription 3 (STAT3), and nuclear factor-kappaB (NF-κB). Experimental validation found that FHF-treated vaginitis rats exhibited reduced intracellular AKT1, STAT3, and NF-κB protein expressions.
Conclusion: Overall, we identified the bioactive compounds and pharmacological mechanisms of FHF against vaginitis, thus offering a theoretical fundament for exploring FHF for treating vaginitis in the future.
Background: Aspiration pneumonia is a severe health concern, particularly for ICU patients with impaired airway defenses. Current animal models fail to fully replicate the condition, focusing solely on chemical lung injury from gastric acid while neglecting pathogen-induced inflammation. This gap hinders research on pathogenesis and treatment, creating an urgent need for a clinically relevant model. This study aimed to develop an improved rat model of aspiration pneumonia by combining hydrochloric acid (HCl) and lipopolysaccharide (LPS) administration.
Methods: Specific pathogen-free Sprague Dawley rats underwent intratracheal instillation of HCl and LPS. Techniques included rat weight measurement, tracheal intubation, pulmonary function monitoring, lung tissue sampling with HE staining and scoring, bronchoalveolar lavage fluid (BALF) sampling, protein and inflammatory cytokine analysis via BCA and ELISA, BALF pH determination, Evans Blue dye assessment, blood gas analysis, FITC-dextran leakage, Western blotting, electron microscopy, survival analysis, and transcriptome sequencing with bioinformatics. Statistical analysis was performed using GraphPad Prism.
Results: The optimal model involved instillation of 1.5 μL/g.wt HCl (pH = 1) followed by 20 μg/g.wt LPS after 1 h. This model reproduced acute lung injury, including tissue damage, pulmonary microvascular dysfunction, inflammatory responses, hypoxemia, and impaired pulmonary ventilation, with recovery observed at 72 h. PANoptosis was confirmed, characterized by increased markers. Concentration-dependent effects of HCl and LPS on lung damage were identified, alongside cytokine elevation and microvascular dysfunction.
Conclusions: This optimized model closely mimics clinical aspiration pneumonia, providing a valuable tool for studying pathophysiology and therapeutic strategies.
Background: Scleral fixation of intraocular lenses is a surgical technique that involves anchoring an artificial lens to the sclera. Traditional approaches, such as capsular bag placement, may not be feasible in certain situations, making scleral fixation a valuable alternative. The scleral reactions to different types of suture materials are not fully understood. Therefore, the present study describes the microscopic structure of normal scleral tissue and its changes with suture materials.
Methods: We compared six groups of rabbit eyes focusing on the sclera: group with polytetrafluoroethylene (PTFE) chain, PTFE fiber, polypropylene (PPE) fiber and control groups. multilevel sampling and stereological methods were used for histological quantification of the leukocyte infiltration fractions and type I and type III collagen.
Results: Quantitative histological evaluation revealed the following: (1) For all materials used, inflammation was present in the surrounding scleral tissue compared with healthy controls. However, leukocyte infiltration in the sclera was not statistically different between the materials. (2) As part of the evaluation of collagen, the greatest changes occurred in the PTFE fiber group at 2 weeks postoperatively. In the PTFE chain group, more significant changes were visible at 4 weeks. (3) The changes in the PPE fiber group compared to healthy scleral tissue were the least significant.
Conclusions: From a histological point of view, it is evident that there are differences in the quantitative parameters between the untouched sclera and the sclera with suture material. Furthermore, distinctions were observed among various materials and across different time intervals.
The Leprdb/db mouse is a common and well-studied model of type II diabetes mellitus that is often employed in biomedical research. Despite being one of the most commonly used models for the investigation of diabetic wound healing, there are a few specific guidelines for its husbandry, and wound complications such as infection and expansion are common. This study presents a modified animal husbandry approach for the Leprdb/db mouse to reduce the incidence of complications during wound healing experiments. Compared to standard rodent housing protocols, the use of this modified protocol leads to decreased rates of complications among experimental animals across several experiments. The protocol includes increased cage size, decreased housing density, and more frequent cage replacements. The use of improved husbandry for the Leprdb/db mouse decreases the total number of animals required, minimizes harm during experimentation, and improves the consistency and reproducibility of wound healing studies.
Sydenham chorea (SC) is the neurological manifestation associated with acute rheumatic fever (ARF). ARF and rheumatic heart disease (RHD) are autoimmune complications triggered by a group A streptococcal (GAS) infection. In ARF/RHD and SC, tissue cross-reactive antibodies and T-cells generated against GAS antigens have been implicated in the pathogenesis. In SC, antibodies against GAS antigens are known to cross-react with neuronal proteins causing neurological manifestations including choreiform movements and neuropsychiatric symptoms such as irritability, attention deficit, and obsessive-compulsive disorder. Previous studies in a rat autoimmune valvulitis (RAV) model of RHD, have shown that injection of streptococcal M protein could cause both cardiac and neurological symptoms. In this study it was shown that adoptive transfer of serum with anti-GAS M antibodies to naive rats caused carditis but failed to demonstrate neurobehavioral symptoms. However, when the blood–brain barrier (BBB) was disrupted using lipopolysaccharide, all animals that received anti-GAS M protein antibodies, developed neurobehavioral defects in addition to carditis. This highlights that impaired BBB integrity is essential for the development of neurobehavioral symptoms. The use of the RAV model and the disruption of BBB required for the development of neurobehavioral changes provides a platform to further investigate the mechanisms that lead to antibodies binding to basal ganglia structures that cause SC.
Murine subarachnoid hemorrhage (SAH) induced using the filament perforation method is a useful in vivo experimental model to investigate the pathophysiological mechanisms in the brain underlying SAH. However, identifying mice with comorbid acute neurogenic pulmonary edema (NPE), a life-threatening systemic consequence often induced by SAH, in this model is difficult without histopathological investigations. Herein, we present an imaging procedure involving dual-energy X-ray absorptiometry (DXA) to identify NPE in a murine model of SAH. We quantified the lung lean mass (LM) and compared the relationship between micro-computed tomography (CT) evidence of Hounsfield unit (HU) values and histopathological findings of PE. Of the 85 mice with successful induction of SAH by filament perforation, 16 (19%) had NPE, as verified by postmortem histology. The DXA-LM values correlate well with CT-HU levels (r = 0.63, p < 0.0001). Regarding the relationship between LM and HU in mice with post-SAH NPE, the LM was positively associated with HU values (r2 = 0.43; p = 0.0056). A receiver operating characteristics curve of LM revealed a sensitivity of 87% and specificity of 57% for detecting PE, with a similar area under the curve as the HU (0.79 ± 0.06 vs. 0.84 ± 0.07; p = 0.21). These data suggest that confirming acute NPE using DXA-LM is a valuable method for selecting a clinically relevant murine NPE model that could be used in future experimental SAH studies.