Dysregulated long noncoding RNAs in the brainstem of the DBA/1 mouse model of SUDEP.

BACKGROUND: Long noncoding RNAs (lncRNAs) play an important role in many neurological diseases. This study aimed to investigate differentially expressed lncRNAs and messenger RNAs (mRNAs) in the susceptibility gaining process of primed DBA/1 mice, a sudden unexpected death in epilepsy (SUDEP) model, to illustrate the potential role of lncRNAs in SUDEP. METHODS: The Arraystar mouse lncRNA Microarray V3.0 (Arraystar, Rockville, MD) was applied to identify the aberrantly expressed lncRNAs and mRNAs between primed DBA/1 mice and normal controls. The differences were verified by qRT-PCR. We conducted gene ontology (GO), the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and coexpression analyses to explore the possible function of the dysregulated RNAs. RESULTS: A total of 502 lncRNAs (126 upregulated and 376 downregulated lncRNAs) and 263 mRNAs (141 upregulated and 122 downregulated mRNAs) were dysregulated with P < 0.05 and a fold change over 1.5, among which Adora3 and Gstt4 were possibly related to SUDEP. GO analysis revealed that chaperone cofactor-dependent protein refolding and misfolded protein binding were among the top ten downregulated terms, which pointed to Hspa1a, Hspa2a and their related lncRNAs. KEGG analysis identified 28 upregulated and 10 downregulated pathways. Coexpression analysis showed fifteen dysregulated long intergenic noncoding RNAs (lincRNAs) and three aberrantly expressed antisense lncRNAs, of which AK012034 and NR_040757 are potentially related to SUDEP by regulating LMNB2 and ITPR1, respectively. CONCLUSIONS: LncRNAs and their coexpression mRNAs are dysregulated in the priming process of DBA/1 in the brainstem. Some of these mRNAs and lncRNAs may be related to SUDEP, including Adora3, Lmnb2, Hspa1a, Hspa1b, Itrp1, Gstt4 and their related lncRNAs. Further study on the mechanism of lncRNAs in SUDEP is needed.

DBA/1 mice display equivalent cardiac function to C57BL/6J mice.

NEW FINDINGS: What is the central question of this study? Do normal adult DBA/1 mice have cardiac function and performance equal to those of C57BL/6J mice? What is the main finding and its importance? Male adult DBA/1 mice show equivalent cardiac function to C57BL/6J mice up to 8 months old. Therefore, cardiac dysfunction could be investigated in an autoimmune diseases model established with DBA/1 mice. ABSTRACT: Cardiovascular mortality has been increasing, and in particular, cardiovascular damage caused by some chronic autoimmune diseases accounts for a large proportion of this. C57BL/6J mice have been used mostly in studies of cardiovascular diseases. However, for purposes of modelling, this strain of mouse has a very low incidence of some chronic immune diseases such as rheumatoid arthritis, to which instead DBA/1 mice are more susceptible. Basic cardiac function differs between mice with different genetic backgrounds. Therefore, we monitored cardiac function and structure of normal male C57BL/6J and DBA/1 mice for six consecutive months. Echocardiography was used to monitor cardiac functions once a month and cardiac systolic function was measured upon isoproterenol challenge at the end of observation. The Excitation-contraction coupling-related proteins were measured by western blotting. Heart tissue sections were subject to haematoxylin-eosin, TUNEL and Alizarin red staining. The results demonstrated that systolic and diastolic function did not vary significantly and both strains were indistinguishable in appearance and structure of hearts. DBA/1 mice showed a good cardiac ß-adrenergic response comparable to C57BL/6J mice with isoproterenol treatment. The phosphorylation of phospholamban at either its protein kinase A or its Ca2+ /calmodulin-dependent protein kinase II site, as well as the activation of troponin I showed no significant difference between strains. These findings suggested that there was no obvious difference in the heart structure and function of normal male DBA/1 mice compared with C57BL/6J mice. The DBA/1 mouse is a strain applicable to investigating autoimmune disease-induced heart dysfunction and exploring potential interventions.

Impact of collagen-induced arthritis on the pharmacokinetic disposition of voriconazole, a widely used antifungal agent: in vitro and in vivo investigations in DBA/1J mice.

Pharmacokinetics of voriconazole, an anti-fungal agent, was determined in collagen-induced arthritic (CIA) and healthy DBA/1J mice. CIA was confirmed in DBA/1J mice by clinical scoring and histological analysis. In vivo oral pharmacokinetic study (3 mg/kg) and in vitro stability assessment in liver microsomes were performed in CIA vs. healthy DBA/1J mice. Additionally, hepatic portal vein cannulated (HPVC) CIA and healthy mice were used to clarify the role of gut first-pass effect. Voriconazole/N-oxide metabolite was measured in plasma and in vitro samples using liquid chromatography tandem-mass spectrometry method. Voriconazole exposure was reduced in CIA by 27% as compared to healthy mice. Formation of voriconazole N-oxide was higher in CIA mice as evidenced by higher molar Cmax ratio (i.e. metabolite/parent) of 2.08 vs. 1.66 in healthy mice. Because voriconazole was stable in microsomes, involvement of presystemic gut metabolism was suspected for decreased voriconazole exposure and formation of higher molar ratio of metabolite. HPVC work revealed higher formation of voriconazole N-oxide in CIA relative to healthy mice resulting in Cmax/AUC ratios of 0.41/0.54 and 0.08/0.17, respectively, confirming first-pass effect. The findings may have implications in the clinical therapy of arthritis patients who are concomitantly given voriconazole for the management of fungal infections.

Hyperimmune colostrum alleviates rheumatoid arthritis in a collagen-induced arthritis murine model.

Our aging population and the accompanying decline in immune function is a growing concern that may be addressed by finding natural methods to enhance the immunocompetence of our elderly. Bovine milk and colostrum from cows that have been immunized have been shown to provide additional immunoglobulins and other bioactive molecules that enhance immune function. The purpose of this study was to investigate the ability of hyperimmune bovine colostrum to alleviate the symptoms of rheumatoid arthritis in a murine model. The collagen-induced arthritis DBA/1J murine model was used for this study. Mice were fed colostrum from immunized cows at either 5 or 10 mg/mouse per day or controls for 49 d. The data showed that the colostrum-fed groups had significantly lower total swelling scores and significantly lower collagen-specific antibody (IgG2a), inflammation-associated antibody (total IgG), and the inflammatory cytokines tumor necrosis factor α, IL-2, IL-6, and IFN-γ. The results strongly suggest that colostrum from immunized cows may have anti-inflammatory activity in a mouse model of rheumatoid arthritis.

Positive selection of type II collagen-reactive CD80high marginal zone B cells in DBA/1 mice.

To investigate whether dysregulated selection of autoreactive marginal zone (MZ) B cells is involved in autoimmune diseases, we examined MZ B cell profile in multiple strains of mice, and found that type II collagen (CII)-reactive autoreactive CD80high MZ B cells spontaneously developed in the DBA/1, but not in C57BL/6 mice. CD80high MZ B cells that were characteristically found in DBA/1 mice expressed higher levels of TACI, SLAM3, and SLAM6 than the usual CD80low MZ B cells. Notably, the CD80high MZ B cells were more sensitive to ibrutinib, a Bruton's tyrosine kinase inhibitor, than CD80low MZ or follicular B cells and their transient depletion via intravenous injection of ibrutinib significantly delayed the induction of collagen-induced arthritis (CIA). In summary, we suggest that the positive selection of CII-reactive CD80high MZ B cells is a critical homeostatic process predisposing the DBA/1 mice to the CIA induction.

Eye-mediated immune tolerance to Type II collagen in arthritis-prone strains of mice.

Type II collagen (CII) is a cartilage structural protein that plays important roles in joint function, arthritis and ageing. In studying the ability of CII to induce eye-mediated specific immune tolerance, we have recently proven that CII is capable of inducing anterior chamber-associated immune deviation (ACAID) in Balb/c mice. Here, we study the ability of CII to induce eye-mediated immune tolerance in strains of mice that are prone to the induction of rheumatoid arthritis. Thus, we hypothesized that CII induces ACAID in DBA/1 mice and in C57BL/6 mice through the AC route (direct injection) or the intravenous route (adoptive transfer of in vitro-generated CII-specific ACAID macrophages or of CII-specific in vitro-generated T regulatory cells). Specific immune tolerance induction was assessed using both delayed-type hypersensitivity (DTH) and local adoptive transfer (LAT) assays. Results indicated the ability of CII to generate CII-specific ACAID-mediated immune tolerance in vivo and in vitro in both DBA/1 mice and C57BL/6 mice. These findings could be beneficial in studies of immune tolerance induction using CII.

Identification of citrullinated α1-antitrypsin (A1AT) in saliva in a mouse model of rheumatoid arthritis.

OBJECTIVES: Rheumatoid arthritis (RA) is an autoimmune disease characterized by progressive joint destruction. Early diagnosis and treatment, before joint deformation or destruction occurs, are crucial. Identifying novel biomarkers for RA in saliva could potentially enable early detection of the disease, prior to its onset. METHODS: We conducted a comprehensive proteomic analysis of salivary proteins in a mouse model of RA. Proteins were identified using western blotting and enzyme-linked immunosorbent assay in the serum, saliva, and ankle joints of DBA/1JJmsSlc mice, a model of RA. Ankle joints and submandibular glands were stained with hematoxylin and eosin and immunostained, and the results were compared with those of control mice. RESULTS: Citrullinated alpha-1 antitrypsin (A1AT, 46 kDa) was commonly detected in the saliva, serum, and ankle joints of mice with severe RA and was confirmed through proteomic analysis. Western blotting showed a band corresponding to 46 kDa in the serum, saliva, and ankle joints. Immunostaining of the ankle joints with the A1AT antibody showed a strong positive signal in the synovium. CONCLUSIONS: In DBA/1JJmsSlc mice, cyclic citrullinated peptide antibodies and A1AT may be involved in citrullination and contribute to the development and severity of RA, making them valuable treatment targets requiring further study.

Disruption of Synaptic Transmission in the Bed Nucleus of the Stria Terminalis Reduces Seizure-Induced Death in DBA/1 Mice and Alters Brainstem E/I Balance.

Sudden unexpected death in epilepsy (SUDEP) is the leading cause of death in refractory epilepsy patients. Accumulating evidence from recent human studies and animal models suggests that seizure-related respiratory arrest may be important for initiating cardiorespiratory arrest and death. Prior evidence suggests that apnea onset can coincide with seizure spread to the amygdala and that stimulation of the amygdala can reliably induce apneas in epilepsy patients, potentially implicating amygdalar regions in seizure-related respiratory arrest and subsequent postictal hypoventilation and cardiorespiratory death. This study aimed to determine if an extended amygdalar structure, the dorsal bed nucleus of the stria terminalis (dBNST), is involved in seizure-induced respiratory arrest (S-IRA) and death using DBA/1 mice, a mouse strain which has audiogenic seizures (AGS) and a high incidence of postictal respiratory arrest and death. The presence of S-IRA significantly increased c-Fos expression in the dBNST of DBA/1 mice. Furthermore, disruption of synaptic output from the dBNST via viral-induced tetanus neurotoxin (TeNT) significantly improved survival following S-IRA in DBA/1 mice without affecting baseline breathing or hypercapnic (HCVR) and hypoxic ventilatory response (HVR). This disruption in the dBNST resulted in changes to the balance of excitatory/inhibitory (E/I) synaptic events in the downstream brainstem regions of the lateral parabrachial nucleus (PBN) and the periaqueductal gray (PAG). These findings suggest that the dBNST is a potential subcortical forebrain site necessary for the mediation of S-IRA, potentially through its outputs to brainstem respiratory regions.

A High-Tryptophan Diet Reduces Seizure-Induced Respiratory Arrest and Alters the Gut Microbiota in DBA/1 Mice.

Background and Aims: Central 5-hydroxytryptamine (5-HT) defects are responsible for the occurrence of sudden unexpected death in epilepsy (SUDEP). The DBA/1 mouse is an animal model of SUDEP since the mouse exhibits audiogenic seizure-induced respiratory arrest (S-IRA). The synthesis of central 5-HT is closely related to the gut microbiota. Moreover, emerging studies suggest a possible role for the microbiota in mitigating seizure likelihood. Based on this, we aimed to explore the effect of a high-tryptophan diet (HTD) on SUDEP as well as the synthesis and metabolism of central 5-HT. Furthermore, we investigated the involvement of the gut microbiota in this process. Methods: All DBA/1 mice were subjected to acoustic stimulation to induce seizures. Only those mice that exhibited S-IRA were randomly assigned to the normal diet (ND) group (n = 39) or HTD group (n = 53). After 1 month of dietary intervention, (1) S-IRA rates were evaluated, (2) the concentrations of 5-HT and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) in the plasma and brain were determined by ultra-high-pressure liquid chromatography, and (3) the fecal flora biodiversity and species composition were analyzed by 16S rDNA microbiota profiling. Results: The S-IRA rate in DBA/1 mice was significantly reduced in the HTD group compared with that in the control group. HTD increased the levels of 5-HT and 5-HIAA in both the telencephalon and midbrain. HTD significantly elevated the species richness and diversity of the gut microbiota. Moreover, there was a significant difference in the gut microbiota composition between the two groups, and the intestinal flora was dominated by Proteobacteria and Actinobacteria after HTD. Conclusions: HTD is efficient in lowering S-IRA rates and elevating the central 5-HT level in DBA/1 mice. The gut microbiota was altered after HTD intervention. The significant increase in Proteobacteria and Actinobacteria may be related to the SUDEP-protective effect of HTD. Our findings shed light on a candidate choice of dietary prevention for SUDEP.

High humidity aggravates the severity of arthritis in collagen-induced arthritis mice by upregulating xylitol and L-pyroglutamic acid.

BACKGROUND: Humidity was an unfavorable factor for patients with rheumatoid arthritis (RA). RA disease activity was severe in high humidity conditions. However, there is no evidence to demonstrate the effects of humidity on arthritis in the animal experiments and explore its relevant mechanism. METHODS: Using the DBA/1 mice, this study addressed the effects of a high humidity (80 ± 5%) on arthritis in collagen-induced arthritis (CIA) mice. Then, this study used the gas chromatography-mass spectrometer (GC-MS) to explore alterations in serum metabolome caused by the high humidity. Furthermore, xylitol and L-pyroglutamic acid, which were both significantly upregulated by the high humidity, were selected to further study their effects on arthritis in the CIA mice. RESULTS: The high humidity (80 ± 5%) could aggravate arthritis variables including increasing arthritis score and swelling, serum autoantibodies (anti-COII and anti-CCP), and proinflammatory cytokines (IL-6, IL-17A, and G-CSF). In addition, the high humidity could cause significant alterations in serum metabolome in the CIA mice. Xylitol and L-pyroglutamic acid were the representative serum metabolites that were significantly upregulated by the high humidity. Further experiments demonstrated that the supplementation of 0.4 mg/mL xylitol in drinking water after inducing the CIA model and 2.0 mg/mL in drinking water before inducing the CIA model could both aggravate arthritis in the CIA mice. CONCLUSIONS: These data demonstrated that high humidity was not beneficial for arthritis development and its mechanism might be associated with xylitol and L-pyroglutamic acid.

CXC chemokine receptor 3 antagonist AMG487 shows potent anti-arthritic effects on collagen-induced arthritis by modifying B cell inflammatory profile.

Several studies have suggested that chemokine receptors are important mediators of inflammatory response in rheumatoid arthritis (RA). B cells are also known to play an important role in RA pathology. C-X-C chemokine receptor type 3 (CXCR3) is considered a potential therapeutic target in different inflammatory diseases; however, the mechanism remains unclear. Here, we evaluated the potentially protective effect of AMG487, a selective CXCR3 antagonist, in collagen-induced arthritis (CIA) mouse model. CIA mice were treated with AMG487 (5 mg/kg) every 48 h, from day 21 until day 41. We then investigated the effect of AMG487 on NF-κB p65-, NOS2-, MCP-1-, TNF-α-, IFN-γ, IL-4-, and IL-27-producing CD19+ B cells in the spleen through flow cytometry. We also evaluated the mRNA and protein expression levels of these molecules using RT-PCR and western blotting in the knee tissues. Our results revealed that AMG487-treated mice showed decreased NF-κB p65-, NOS2-, MCP-1-, and TNF-α-, and increased IL-4-, and IL-27-producing CD19+ B cells compared with the control mice. Additionally, AMG487 treatment significantly down regulated NF-κB p65, NOS2, TNF-α, and IFN-γ, and upregulated IL-4 and IL-27 mRNA and protein expression levels compared with the control. Thus, our study shows that AMG487 exerts its anti-arthritic effect by potently downregulating inflammatory B cell signaling. Based on our observations, we propose that AMG487 could serve as a potential novel therapeutic agent for inflammatory and autoimmune diseases, including RA.

CXCR3 antagonist AMG487 suppresses rheumatoid arthritis pathogenesis and progression by shifting the Th17/Treg cell balance.

Rheumatoid arthritis (RA) is an autoimmune disease that is characterized by uncontrolled joint inflammation and damage to bone and cartilage. Previous studies have shown that chemokine receptors have important roles in RA development, and that blocking these receptors effectively inhibits RA progression. Our study was undertaken to investigate the role of AMG487, a selective CXCR3 antagonist, in DBA/1J mice bearing collagen-induced arthritis (CIA). Following induction of CIA, animals were treated with 5 mg/kg AMG487 intraperitoneally every 48 h, starting from day 21 until day 41 and evaluated for clinical score, and histological hallmarks of arthritic inflammation. We further investigated the effect of AMG487 on Th1 (T-bet), Th17 (IL-17A, RORγt, STAT3), Th22 (IL-22), and T regulatory (Treg; Foxp3 and IL-10) cells in splenic CXCR3+ and CD4+ T cells using flow cytometry. We also assessed the effect of AMG487 on T-bet, RORγt, IL-17A, IL-22, Foxp3, and IL-10 at both mRNA and protein levels using RT-PCR and Western blot analyses of knee samples. The severity of clinical scores, and histological inflammatory damage decreased significantly in AMG487-treated compared with CIA control mice. Moreover, the percentage of Th1, Th17, and Th22 cells decreased significantly and that of Treg cells increased in AMG487-treated mice. We further observed that AMG487-treatment downregulated T-bet, IL-17A, RORγt, and IL-22, whereas it upregulated Foxp3 and IL-10 mRNA and protein levels. This study demonstrates the antiarthritic effects of AMG487 in CIA animal model and supports the development of CXCR3 antagonists as a novel strategy for the treatment of inflammatory and arthritic conditions.

Specific subcortical structures are activated during seizure-induced death in a model of sudden unexpected death in epilepsy (SUDEP): A manganese-enhanced magnetic resonance imaging study.

Sudden unexpected death in epilepsy (SUDEP) is a major concern for patients with epilepsy. In most witnessed cases of SUDEP generalized seizures and respiratory failure preceded death, and pre-mortem neuroimaging studies in SUDEP patients observed changes in specific subcortical structures. Our study examined the role of subcortical structures in the DBA/1 mouse model of SUDEP using manganese-enhanced magnetic resonance imaging (MEMRI). These mice exhibit acoustically-evoked generalized seizures leading to seizure-induced respiratory arrest (S-IRA) that results in sudden death unless resuscitation is rapidly instituted. MEMRI data in the DBA/1 mouse brain immediately after acoustically-induced S-IRA were compared to data in C57 (control) mice that were exposed to the same acoustic stimulus that did not trigger seizures. The animals were anesthetized and decapitated immediately after seizure in DBA/1 mice and after an equivalent time in control mice. Comparative T1 weighted MEMRI images were evaluated using a 14T MRI scanner and quantified. We observed significant increases in activity in DBA/1 mice as compared to controls at previously-implicated auditory (superior olivary complex) and sensorimotor-limbic [periaqueductal gray (PAG) and amygdala] networks and also in structures in the respiratory network. The activity at certain raphe nuclei was also increased, suggesting activation of serotonergic mechanisms. These data are consistent with previous findings that enhancing the action of serotonin prevents S-IRA in this SUDEP model. Increased activity in the PAG and the respiratory and raphe nuclei suggest that compensatory mechanisms for apnea may have been activated by S-IRA, but they were not sufficient to prevent death. The present findings indicate that changes induced by S-IRA in specific subcortical structures in DBA/1 mice are consistent with human SUDEP findings. Understanding the changes in brain activity during seizure-induced death in animals may lead to improved approaches directed at prevention of human SUDEP.

Whole mitochondrial genome sequence for an osteoarthritis model of spontaneous mice DBA/1 strain.

Animal models play an important role in osteoarthritis studies. Here, the complete mitochondrial genome sequence of the spontaneous mice DBA/1 strain was reported for the first time. The total length of the mitogenome was 16,769 bp. It contained the typical structure, including two ribosomal RNA genes, 13 protein-coding genes, 22 transfer RNA genes, and one non-coding control region (D-loop region). The overall GC content of the mitogenome was estimated to be 39.2%. This mitochondrial genome sequence will provide new genetic resource into osteoarthritis disease.

Induction of protective immunity against Chlamydia muridarum intracervical infection in DBA/1j mice.

We previously reported that intracervical inoculation with Chlamydia muridarum induced hydrosalpinx in DBA/1j mice, but intravaginal inoculation failed to do so. In the current study, we found unexpectedly that intrabursal inoculation of live chlamydial organisms via the oviduct failed to induce significant hydrosalpinx. We further tested whether primary infection via intravaginal or intrabursal inoculation could induce protective immunity against hydrosalpinx following intracervical challenge infection. Mice infected intravaginally with C. muridarum were fully protected from developing hydrosalpinx, while intrabursal inoculation offered partial protection. We then compared immune responses induced by the two genital tract inoculations. Both inoculations induced high IFNγ and IL-17 T cell responses although the ratio of IgG2a versus IgG1 in intravaginally infected mice was significantly higher than in mice infected intrabursally. When the antigen-specificities of antibody responses were compared, both groups of mice dominantly recognized 24 C. muridarum antigens, while each group preferentially recognized unique sets of antigens. Thus, we have demonstrated that intrabursal inoculation is neither effective for causing hydrosalpinx nor efficient in inducing protective immunity in DBA/1j mice. Intravaginal immunization, in combination with intracervical challenge infection in DBA/1j mice, can be a useful model for understanding mechanisms of chlamydial pathogenicity and protective immunity.