C57BL6J Mice Research Articles

Metabolomic profiling reveals muscle metabolic changes following iliac arteriovenous fistula creation in mice.

End-stage kidney disease, the most advanced stage of chronic kidney disease (CKD), requires renal replacement therapy or kidney transplant to sustain life. To accomplish durable dialysis access, the creation of an arteriovenous fistula (AVF) has emerged as a preferred approach. Unfortunately, a significant proportion of patients that receive an AVF experience some form of hand dysfunction; however, the mechanisms underlying these side effects are not understood. In this study, we used nuclear magnetic resonance spectroscopy to investigate the muscle metabolome following iliac AVF placement in mice with CKD. To induce CKD, C57BL6J mice were fed an adenine-supplemented diet for 3 wk and then randomized to receive AVF or sham surgery. Two weeks following surgery, the quadriceps muscles were rapidly dissected and snap frozen for metabolite extraction and subsequent nuclear magnetic resonance analysis. Principal component analysis demonstrated clear separation between groups, confirming a unique metabolome in mice that received an AVF. AVF creation resulted in reduced levels of creatine, ATP, and AMP as well as increased levels of IMP and several tricarboxylic acid cycle metabolites suggesting profound energetic stress. Pearson correlation and multiple linear regression analyses identified several metabolites that were strongly linked to measures of limb function (grip strength, gait speed, and mitochondrial respiration). In summary, AVF creation generates a unique metabolome profile in the distal skeletal muscle indicative of an energetic crisis and myosteatosis.NEW & NOTEWORTHY Creation of an arteriovenous fistula (AVF) is the preferred approach for dialysis access, but some patients experience hand dysfunction after AVF creation. In this study, we provide a detailed metabolomic analysis of the limb muscle in a murine model of AVF. AVF creation resulted in metabolite changes associated with an energetic crisis and myosteatosis that associated with limb function.

Shibi Tea (Adinandra nitida) and Camellianin A Alleviate CCl4-Induced Liver Injury in C57BL-6J Mice by Attenuation of Oxidative Stress, Inflammation, and Apoptosis.

Liver injury is a significant public health issue nowadays. Shibi tea is a non-Camellia tea prepared from the dried leaves of Adinandra nitida, one of the plants with the greatest flavonoid concentration, with Camellianin A (CA) being the major flavonoid. Shibi tea is extensively used in food and medicine and has been found to provide a variety of health advantages. The benefits of Shibi tea and CA in preventing liver injury have not yet been investigated. The aim of this study was to investigate the hepatoprotective effects of extract of Shibi tea (EST) and CA in mice with carbon tetrachloride (CCl4)-induced acute liver injury. Two different concentrations of EST and CA were given to model mice by gavage for 3 days. Treatment with two concentrations of EST and CA reduced the CCl4-induced elevation of the liver index, liver histopathological injury score, alanine aminotransferase (ALT), and aspartate aminotransferase (AST). Western blotting and immunohistochemical analysis demonstrated that EST and CA regulated the oxidative stress signaling pathway protein levels of nuclear factor E2-related factor 2 (Nrf2)/heme-oxygenase-1 (HO-1), the expression of inflammatory cytokines, the phosphorylated nuclear factor-kappaB p65 (p-NF-κB)/nuclear factor-kappaB p65 (NF-κB) ratio, the phospho-p44/42 mitogen-activated protein kinase (p-MAPK), and the apoptosis-related protein levels of BCL2-associated X (Bax)/B cell leukemia/lymphoma 2 (Bcl2) in the liver. Taken together, EST and CA can protect against CCl4-induced liver injury by exerting antioxidative stress, anti-inflammation, and anti-apoptosis.

The changing landscape of immune cells in the fetal mouse testis.

Fetal testis growth involves cell influx and extensive remodeling. Immediately after sex determination in mouse, macrophages enable normal cord formation and removal of inappropriately positioned cells. This study provides new information about macrophages and other immune cells after cord formation in fetal testes, including their density, distribution, and close cellular contacts. C57BL6J mouse testes from embryonic day (E) 13.5 to birth (post-natal day 0; PND0), were examined using immunofluorescence, immunohistochemistry, and RT-qPCR to identify macrophages (F4/80, CD206, MHCII), T cells (CD3), granulocytes/neutrophils (Ly6G), and germ cells (DDX4). F4/80+ cells were the most abundant, comprising 90% of CD45+ cells at E13.5 and declining to 65% at PND0. Changes in size, shape, and markers (CD206 and MHCII) documented during this interval align with the understanding that F4/80+ cells have different origins during embryonic life. CD3+ cells and F4/80−/MHCII+ were absent to rare until PND0. Ly6G+ cells were scarce at E13.5 but increased robustly by PND0 to represent half of the CD45+ cells. These immunofluorescence data were in accord with transcript analysis, which showed that immune marker mRNAs increased with testis age. F4/80+ and Ly6G+ cells were frequently inside cords adjacent to germ cells at E13.5 and E15.5. F4/80+ cells were often in clusters next to other immune cells. Macrophages inside cords at E13.5 and E15.5 (F4/80Hi/CD206+) were different from macrophages at PND0 (F4/80Dim/CD206−), indicating that they have distinct origins. This histological quantification coupled with transcript information identifies new cellular interactions for immune cells in fetal testis morphogenesis, and highlights new avenues for studies of their functional significance.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00418-022-02129-6.

Abstract 2519: Polymorphonuclear myeloid-derived suppressor cells reflect the status of peritoneal dissemination in colon cancer mouse model

Abstract Background 8.3% of colorectal cancer (CRC) patients have peritoneal dissemination (PD) and their prognosis is poor. Myeloid-derived suppressor cells, MDSC, are functional myeloid cells with immune suppressive properties. When infiltrated into the tumor, these cells induce dysfunction of cytotoxic T cells via the secretion of immunosuppressive molecules like arginase-1 and promote tumor progression. MDSC is classified into two major subsets in a mouse by their surface antigens: monocytic MDSCs (M-MDSCs; CD11b+Ly6ChighLy6G−) and polymorphonuclear MDSCs (PMN-MDSCs; CD11b+Ly6ClowLy6G+). To date, MDSCs are difficult to identify because they express the same surface markers as neutrophils and monocytes, respectively. CD244, an immunoregulatory transmembrane receptor molecule, has been highlighted as a surface antigen to distinguish PMN-MDSCs from normal neutrophils. However, few studies have demonstrated whether and how MDSCs, especially CD244+ PMN-MDSC, affect the generation and progression of PD. Objective To clarify CD244+ PMN-MDSC affects the progression of PD and CD8+ T cells. Methods Female C57BL6J mice were injected 5.0×105 ~ 2.0×106 cells of MC38 colon cancer intraperitoneally to establish the PD model, and we had conducted the following three experiments: Exp. 1) Investigation of the trend of immune cells including MDSC and CD8+ T cell in the PD nodule, peritoneal cavity, blood, and spleen; Exp. 2) PMN-MDSC depletion test in PD model; and Exp. 3) Investigation of direct influence of CD244+ or CD244- Ly6G+ cells in T cell proliferation in vitro. Results Exp. 1) PD nodules became enlarged and increased over time. All mice had died by day 40 after the cancer was inoculated in the PD model, which was significantly shorter than the MC38-based subcutaneously inoculation model. In the PD model, a significant increase of PMN-MDSCs was observed in the peritoneal cavity, blood, and spleen, and also observed these cells in the PD nodule at day 20. Of note, the CD244+ PMN-MDSC predominantly existed in the population of myeloid cells in the peritoneal cavity. Exp. 2) To deplete the PMN-MDSC, the mice were administered a Ly6G antibody. Although Ly6G treatment could not improve the survival rate of the PD model, the tumor progression was significantly decreased, and the number of both CD4+ T cells and CD8+ T cells was notably increased in the peritoneal cavity at day 19. Exp. 3) Finally, we sorted CD244+/CD244- Ly6G+ cells and co-cultured them with splenocytes. The Ly6G+CD244+ cells significantly inhibited antigen-specific CD8+ T cell proliferation in a dose-dependent manner. T cell suppression mediated by the Ly6G+CD244+ cells was also observed in the numerical evaluation of CD8+ T cells at a high (1:1) ratio. Conclusion The targeted therapy for PMN-MDSCs would provide not only new therapeutic value but also a novel strategy to synergize with T cell-based immunotherapy for CRC-derived PD. Citation Format: Masafumi Saito, Yutaka Sugita, Kimihiro Yamashita, Mitsugu Fujita, Kota Yamada, Kyosuke Agawa, Akihiro Watanabe, Eiji Fukuoka, Shingo Kanaji, Taro Oshikiri, Takeru Matsuda, Yoshihiro Kakeji. Polymorphonuclear myeloid-derived suppressor cells reflect the status of peritoneal dissemination in colon cancer mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2519.

Polystyrene nanoparticles aggravate the adverse effects of di-(2-ethylhexyl) phthalate on different segments of intestine in mice

Emerging evidence indicates that nanoplastics (NPs) can transport organic pollutants such as di-(2-ethylhexyl) phthalate (DEHP) into organisms and induce adverse health effects. Nevertheless, the toxic effects of NPs combined with DEHP on mammalian intestine are still unclear. In this study, the C57BL6J mice were exposed to polystyrene nanoparticles (PSNPs), DEHP or them both for 30 days to determine their effects on different segments of intestine and the gut microbiota. As a result, DEHP alone or co-exposure to DEHP and PSNPs induced histological damages in all intestinal parts, mainly manifested as the decreased villus lengths, increased crypt depths in the duodenum, jejunum and ileum and decreased villus counts accompanied with decreased epithelial area in the colon. Moreover, decreased mucus coverage, down-regulated Muc2 expression levels as well as the broken tight junctions were observed in intestinal epithelium of mice, particularly obvious in the co-treatment groups. In general, as manifested by greater alterations in most of the parameters mentioned above, simultaneously exposed to PSNPs and DEHP seemed to induce enhanced toxic effects on intestine of mouse when compared with DEHP alone. Furthermore, the altered community composition of gut microbiota might at least partially contribute to these abnormalities. Overall, our results highlight the aggravated toxicity on different segments of intestine in mammalians due to co-exposure of PSNPs and DEHP, and these findings will provide valuable insights into the health risk of NPs and plastic additives.

A Metabolomics Approach to Sulforaphane Efficacy in Secondhand Smoking-Induced Pulmonary Damage in Mice.

Sulforaphane is an isocyanate abundantly present in cruciferous vegetables. In the present study, we aimed to investigate the effects of sulforaphane on secondhand smoking (SHS)-induced pulmonary damage in mice. Additionally, a metabolomic study was performed to identify biomarkers associated with pulmonary disease using proton nuclear magnetic resonance (1H-NMR) analysis. Male C57BL6J mice were divided into a control group, an SHS exposure group (positive control group, PC), and a sulforaphane treatment group exposed to secondhand smoke (SS) (n = 5 per group). The PC and SS groups were exposed to secondhand smoke in a chamber twice daily for four weeks. Mice in the SS group were orally administered sulforaphane (50 mg/kg) for four weeks during secondhand smoke exposure. Histopathological examination of the lungs revealed pulmonary damage in PC mice, including loss of bronchial epithelial cells, bronchial wall thickening, and infiltration of macrophages. In contrast, mice in the SS group showed little or no epithelial thickening, thereby exhibiting reduced lung damage. Mouse serum and lung tissues were collected and analyzed to determine changes in endogenous metabolites using 1H-NMR. After target profiling, we identified metabolites showing the same tendency in the serum and lung as biomarkers for SHS-induced pulmonary damage, including taurine, glycerol, creatine, arginine, and leucine. As a result of histopathological examination, sulforaphane might inhibit SHS-induced lung damage, and metabolite analysis results suggest potential biomarkers for SHS-induced pulmonary damage in mice.

Ecballium elaterium improved stimulatory effects of tissue-resident NK cells and ameliorated liver fibrosis in a thioacetamide mice model.

Ecballium elaterium (EE), widely used plant in Mediterranean medicine, showed anticancer activity. This study aimed to investigate EE effects on liver fibrosis in an animal model of thioacetamide (TAA). Intraperitoneal administration of TAA was performed twice weekly for four weeks in C57BL6J mice. Livers were extracted and serum were evaluated for inflammatory markers (H&E staining, ALT, AST, ALP), pro-inflammatory cytokines, fibrosis (Sirius red staining, Masson's trichrome, α-smooth muscle actin and collagen III), and metabolic (cholesterol, triglyceride, C-peptide, and fasting-blood-sugar) profiles. Glutathione, glutathione peroxidase, and catalase liver antioxidant markers were assessed. Tissue-resident NK cells from mice livers were functionally assessed for activating receptors and cytotoxicity. Compared to vehicle-treated mice, the TAA-induced liver injury showed attenuation in the histopathology outcome following EE treatment. In addition, EE-treated mice resulted in decreased serum levels of ALT, AST, and ALP, associated with a decrease in IL-20, TGF-β, IL-17, IL-22 and MCP-1 concentrations. Moreover, EE-treated mice exhibited improved lipid profile of cholesterol, triglycerides, C-peptide, and FBS. EE treatment maintained GSH, GPX, and CAT liver antioxidant activity and led to elevated counts of tissue-resident NK (trNK) cells in the TAA-mice. Consequently, trNK demonstrated an increase in CD107a and IFN-γ with improved potentials to kill activated hepatic-stellate cells in an in vitro assay. EE exhibited antifibrotic and antioxidative effects, increased the number of trNK cells, and improved metabolic outcomes. This plant extract could be a targeted therapy for patients with advanced liver injury.

Ticagrelor Protects against Sepsis-Induced Acute Kidney Injury through an Adenosine Receptor-Dependent Pathway.

Ticagrelor is a widely used anti-platelet drug. However, the mechanisms by which ticagrelor protects against sepsis-induced acute kidney injury (AKI) have not been clearly demonstrated. We designed this study to explore the protective effect of ticagrelor on sepsis-induced AKI and to explore the underlying mechanisms. C57BL6J mice received oral ticagrelor (20 mg/kg and 50 mg/kg) for 7 days, and then caecal ligation and puncture (CLP) were performed. An adenosine receptor antagonist, CGS15943, was administered (10 mg/kg, intraperitoneal injection) to block the adenosine pathway 2 h before CLP. After 24 h, serum creatinine levels were measured. Periodic acid-Schiff (PAS) staining and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining were employed to analyze pathological changes and cell apoptosis. Serum concentrations of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and mRNA expression of tissue TNF-α and IL-1β were detected. Western blotting analysis was used to determine AKT and mammalian target of rapamycin (mTOR) protein expression in the kidney. PAS staining showed less swelling of renal tubules, and TUNEL staining revealed less cell apoptosis in the ticagrelor group than in the CLP group. Serum creatinine levels were significantly lower in the ticagrelor group than in the CLP group. Moreover, significantly lower serum and kidney levels of TNF-α and IL-1β were observed in the ticagrelor group. CGS15943 blocked the effects of ticagrelor. Western blotting analysis showed increased phosphorylation of AKT and mTOR in the kidneys of the 50 mg/kg ticagrelor group. The adenosine receptor antagonist inhibited the activation of AKT and mTOR. This study demonstrates that the protective effect of ticagrelor on sepsis-induced AKI depends on adenosine receptor activation and the subsequent increase of AKT and mTOR phosphorylation.

1337-P: Sex-Specific Variation in Lipid Metabolism in Response to Nutritional Stress Is Associated with Differences in NASH Progression

Nonalcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease in the US, encompassing a disease spectrum defined by steatosis, inflammation (NASH) , and fibrosis. Although the incidence of NASH is less in women compared with men, the mechanistic basis for this phenomenon is unclear. The aim of this study was to use high-resolution LC-MS/MS proteomics to determine sex-specific differences in liver protein abundance in healthy mice and in response to NASH that may contribute to protection in females. Male and female C57BL6J mice were fed regular chow (RC) or fast food (FF) diet (40% fat, 0.15% cholesterol, 42 g/L high-fructose corn syrup drinking water; n=18-20/g) to induce NASH. Liver weight and steatosis were significantly increased in male and female FF mice compared to RC counterparts, and the effects were more pronounced in males versus females. Plasma insulin and ALT followed similar patterns. Label-free differential mass spectrometry identified 2,917 proteins, where 314 and 445 proteins exhibited statistically significant changes in expression associated with sex and diet, and 2proteins showed a significant sex x diet interaction. Unsupervised hierarchical clustering of the 2proteins identified four groups of proteins defined by sex-specific differences in baseline abundance or response to FF-diet. Enrichment analysis of proteins within the four groups identified biological process related to 1) lipid catabolism and synthesis that were upregulated in FF males compared with females; 2) catabolism and oxidation-reduction reactions that were suppressed by FF diet in males but not females and 3) acyl-CoA hydrolase activity and fatty acid oxidation that were higher in RC females than males and induced by FF in males but not females. These data suggest that sex-specific differences in liver lipid metabolism at baseline and in response to nutritional stress may contribute to sexual dimorphism in NASH susceptibility. Disclosure M.Moon: None. A.Murali: None. F.Bello: None. J.Wang: None. M.J.Jurczak: None. Funding National Institutes of Health (DK114012)

Evaluation of Recombinant Botulinum Neurotoxin Type A1 Efficacy in Peripheral Inflammatory Pain in Mice.

Well-established efficacy of botulinum neurotoxin type A (BoNT/A) in aesthetic dermatology and neuromuscular hyperactivity disorders relies on canonical interruption of acetylcholine neurotransmission at the neuromuscular junction at the site of the injection. The mechanisms and the site of activity of BoNT/A in pain, on the other hand, remain elusive. Here, we explored analgesic activity of recombinant BoNT/A1 (rBoNT/A1; IPN10260) in a mouse model of inflammatory pain to investigate the potential role of peripheral sensory afferents in this activity. After confirming analgesic efficacy of rBoNT/A1 on CFA-induced mechanical hypersensitivity in C57Bl6J mice, we used GCaMP6s to perform in vivo calcium imaging in the ipsilateral dorsal root ganglion (DRG) neurons in rBoNT/A1 vs. vehicle-treated mice at baseline and following administration of a range of mechanical and thermal stimuli. Additionally, immunohisochemical studies were performed to detect cleaved SNAP25 in the skin, DRGs and the spinal cord. Injection of CFA resulted in reduced mechanical sensitivity threshold and increased calcium fluctuations in the DRG neurons. While rBoNT/A1 reduced mechanical hypersensitivity, calcium fluctuations in the DRG of rBoNT/A1- and vehicle-treated animals were similar. Cleaved SNAP25 was largely absent in the skin and the DRG but present in the lumbar spinal cord of rBoNT/A1-treated animals. Taken together, rBoNT/A1 ameliorates mechanical hypersensitivity related to inflammation, while the signal transmission from the peripheral sensory afferents to the DRG remained unchanged. This strengthens the possibility that spinal, rather than peripheral, mechanisms play a role in the mediation of analgesic efficacy of BoNT/A in inflammatory pain.

Astragaloside IV Ameliorates Isoprenaline-Induced Cardiac Fibrosis in Mice via Modulating Gut Microbiota and Fecal Metabolites.

AimGut microbiota is of crucial importance to cardiac health. Astragaloside IV (AS-IV) is a main active ingredient of Huangqi, a traditional edible and medicinal herb that has been shown to have beneficial effects on cardiac fibrosis (CF). However, it is still uncertain whether the consumption of AS-IV alleviates cardiac fibrosis through the gut microbiota and its metabolites. Therefore, we assessed whether the anti-fibrosis effect of AS-IV is associated with changes in intestinal microbiota and fecal metabolites and if so, whether some specific gut microbes are conducive to the benefits of AS-IV.MethodsMale C57BL-6J mice were subcutaneously injected with isoprenaline (ISO) to induce cardiac fibrosis. AS-IV was administered to mice by gavage for 14 days. The effects of AS-IV on cardiac function, myocardial enzyme, cardiac weight index (CWI), and histopathology of ISO-induced CF mice were investigated. Moreover, 16S rRNA sequencing was used to establish gut-microbiota profiles. Fecal-metabolites profiles were established using the liquid chromatograph-mass spectrometry (LC-MS).ResultsAS-IV treatment prevented cardiac dysfunction, ameliorated myocardial damage, histopathological changes, and cardiac fibrosis induced by ISO. AS-IV consumption increased the richness of Akkermansia, Defluviitaleaceae_UCG-011, and Rikenella. AS-IV also modulated gut metabolites in their feces. Among 141 altered gut metabolites, amino acid production was sharply changed. Furthermore, noticeable correlations were found between several specific gut microbes and altered fecal metabolites.ConclusionsAn increase of Akkermansia, Defluviitaleaceae_UCG-011, and Rikenella abundance, and modulation of amino acid metabolism, may contribute to the anti-fibrosis and cardiac protective effects of Astragaloside IV.

Abdominal Aortic Aneurysm and Plasminogen Activator Inhibitor 1: Correlation in the Context of Type 2 Diabetes

BackgroundAbdominal Aortic Aneurysm (AAA) is a progressive, inflammatory disease of the infrarenal aorta, characterized by irreversible aortic dilation. Despite decades of research, AAA pathogenesis remains poorly understood, and no pharmaceutical therapies to slow aortic growth have been identified. Interestingly, the only condition known to be associated with decreased rates of aortic growth and rupture in AAA patients is a diagnosis of Type 2 Diabetes Mellitus (T2DM). Our laboratory has demonstrated that streptozotocin (STZ)‐induced hyperglycemia inhibits aortic dilation in a mouse model of AAA. Plasminogen Activator Inhibitor 1 (PAI‐1), a key player in the fibrinolytic pathway, has been shown to be elevated in both Type 1 and T2DM patients and hyperglycemic mice. Additionally, studies have demonstrated that local application of PAI‐1 is effective in inhibiting AAA development in a murine model.ObjectiveThe purpose of the present study was to determine the role of PAI‐1 expression in hyperglycemic protection against AAA growth.HypothesisWe predicted that elevated PAI‐1 is responsible for decreased fibrinolysis leading to inhibited aortic growth in patients with both diabetes and AAA. Based on this hypothesis, we proposed that hyperglycemic mice deficient in PAI‐1 (PAI‐1 KO) would not demonstrate the protection from AAA growth observed in hyperglycemic C57BL/6J (C57) control mice.MethodsFasting blood glucose (BG; mg/dL) was measured in 6‐week‐old C57 and PAI‐1 KO mice at baseline on experimental day (D)0, and STZ (40 mg/kg) was intraperitoneally administered daily from D0 to D4. Sodium citrate buffer (SC; vehicle) was administered to control mice. On D14, final BG was measured to confirm hyperglycemia (≥250 mg/dL). On D21, mice underwent laparotomy: a midline abdominal incision was made and the infrarenal aorta was isolated. After measuring aortic diameter midway between the renal arteries and iliac bifurcation, pancreatic porcine elastase (PPE; 60 U/mL) was applied to the adventitial aorta for 5 minutes. The aorta and surrounding structures were then washed twice with 0.9% sterile saline. Control mice received heat‐inactivated PPE. On D49, the incision was reopened, and the aorta was isolated and measured at the widest point of dilation.ResultsSTZ‐treated C57 mice experienced significantly reduced AAA diameter compared to SC‐treated C57 controls (p< 0.0001) 4 weeks post‐induction. However, differences in AAA diameter were not observed under the same treatment conditions in PAI‐1 KO mice (p= 0.290). Percent increase in aortic diameter in both STZ‐ and SC‐treated PAI‐1 KO mice induced with AAA was significantly reduced compared to that of SC‐treated C57 mice (p< 0.01 and p < 0.0001, respectively).ConclusionsOur study demonstrated that PAI‐1 is elevated in mice with STZ‐induced diabetes. While hyperglycemic mice show significantly reduced AAA growth compared to normoglycemic AAA controls, no difference in aortic growth is observed between hyperglycemic and normoglycemic PAI‐1 KO mice. Regardless of treatment status, PAI‐1 deficient mice experience significantly reduced AAA growth compared to C57 control mice. We conclude that while the fibrinolytic pathway and PAI‐1 are influential in AAA development, hyperglycemia‐induced elevated PAI‐1 expression does not play a crucial role in hyperglycemic inhibition of AAA growth. Conversely, PAI‐1 deficiency appears to inhibit AAA growth regardless of glycemic status.

The Impact of HIV Infection in Cocaine Seeking Behaviors

BackgroundSubstance use disorders (SUDs) are characterized by high propensity to relapse and are highly comorbid with HIV infection. The underlying neurobiology of this relationship is poorly understood. Preclinical research on the neurobiobehavioral outcomes of progressive HIV infections may yield crucial information to improve SUD prognosis and reduce risk of relapse in people living with HIV.MethodsTo model progressive HIV, adult male and female C57BL6J mice were inoculated with EcoHIV, a chimeric HIV‐1 which infects murine cells by replacing envelope glycoprotein gp120 with gp80. Five weeks after inoculation mice were trained in a cocaine conditioned place preference (CPP) paradigm (10mg/kg, i.p.) followed by extinction training. Stress‐induced reinstatement was assessed following administration of yohimbine, an α2 adrenergic antagonist, to determine EcoHIV effects. Neuronal activation in the prefrontal cortex (PFC) and nucleus accumbens (NAc) by cocaine exposure was assessed by quantitative analysis the expression of the immediate early gene, cFos.ResultsEcoHIV and sham‐inoculated mice exhibited similar cocaine CPP and extinction. However, EcoHIV mice showed significantly increased yohimbine‐induced reinstatement rate (p<0.05). cFos analysis revealed an elevated neural activation in PFC and NAc in response to cocaine. However, only neurons within infralimbic PFC (IL) and NAc shell subregions showed further increases in activity in cocaine exposed mice infected with EcoHIV vs shams.ConclusionEcoHIV infected mice escalated stress‐induced reinstatement of cocaine seeking compared to controls, suggesting that people living with HIV may be at elevated risk for stress‐induced relapse. Progressive EcoHIV infection may dysregulate IL and NAcShell activity to facilitate the cocaine seeking and reinstatement. Ongoing research will characterize how EcoHIV infection dysregulate IL‐NAcShell pathway specific activity during cocaine reward learning and stress‐induced relapse.

A history of low dose ethanol modulates ventral hippocampus activity during motivated behavior

More than half of the 130 million people in the U.S. who consumed alcohol within the last month did so at a level that did not meet diagnostic criteria for an alcohol binge. However, even these sub diagnostic drinking levels can have neurobehavioral outcomes. Clinical studies have shown that low levels of acute ethanol drinking soon after learning can enhance task consolidation and memory recall. Our preclinical data in C57BL6J mice show that a history of repeated, low dose ethanol (0.5g/kg) exposure increases sucrose reward motivation as measured by progressive ratio. The ventral hippocampus (vHPC) is increasingly considered a critical contributor to reward seeking and has been shown to be sensitive to chronic ethanol exposure at higher doses. The present study sought to determine whether daily, post‐training, low dose ethanol exposure can modulate vHPC activity during motivated behavior. To investigate this, we implanted mice with an electrode array to record vHPC neural activity during performance of a progressive ratio task. We also investigated vHPC activity around important behavioral events in the task, including lever pressing (when the animal is seeking the reward) and magazine entries (when the animal is checking for reward delivery). Our results demonstrate that saline control mice show suppression of vHPC activity following a lever press, while mice with a history of low dose ethanol exposure exhibit this suppression prior to a lever press. In contrast, both saline and ethanol‐exposed mice exhibit similar vHPC activity patterns surrounding magazine entry. These results suggest that a history of post‐training low dose ethanol exposure modulates vHPC activity during reward seeking. Further, this shift in vHPC suppression around a lever press may be associated with the enhanced motivation for sucrose observed in ethanol‐exposed mice. Future studies will use optogenetics to determine whether this shift in activity contributes to heightened reward motivation in ethanol exposed mice. A greater understanding of the neurobiological substrates impacted by ethanol exposure can help identify factors placing casual drinkers at risk for transition to alcohol use disorder.

Mice Exposed to Chronic low‐dose Ethanol Exhibit Reduced Sensitivity to Changes in Magnitude of Reward

While approximately 2 billion individuals consume alcohol worldwide, the majority of these individuals to not exhibit drinking patterns consistent with alcohol use disorders (AUDs). Efforts have been made in investigating and characterizing the adverse effects on learning, memory and reward seeking with individuals afflicted with AUDs in a pursuit of therapeutic advancements. However, sub‐diagnostic alcohol effects have been understudied, despite the large number of low‐dose alcohol drinkers and the potential for low dose alcohol to impact both brain and behavior.Data from our lab has shown that low‐dose alcohol exposure enhances reward motivation in male, but not female, mice, but the mechanism of this is effect is unclear. Thus, we investigated the effects of low‐dose alcohol exposure on sensitivity to change in reward magnitude. A group of adult male C57BL6J mice were trained to self‐administer a 20 ul sucrose reward and received either a low‐dose ethanol (0.5g/kg) or saline injection 1 hour after training. To assess motivation, we used a progressive ratio (PR) task in which the effort required to earn the sucrose reward was increased linearly. Mice received multiple counterbalanced PR sessions in which the progressive schedule remained the same, but the reward magnitude was changed from 20ul (baseline) to 35ul, 25ul, 15ul or 5ul in counterbalanced sessions.Consistent with sensitivity to changes in reward value, saline control mice reduced or increased responding when reward volume was reduced or increased, respectively. Similar to saline controls, chronic low‐dose ethanol exposed mice increased their responding when reward volumes were increased compared to the 20ul baseline. However, ethanol exposed mice did not reduce responding to small reductions in reward magnitude. This observation in low‐dose ethanol mice suggests either a failure to detect reductions in reward magnitude or a dissociation between reduced reward magnitude and behavioral response. This lack of strategizing behavior to maintain effort‐to‐reward balance by ethanol exposed mice may imply that chronic low‐dose ethanol exposed mice may perceive greater reward value than controls even at reduced volume and implicates even low‐dose ethanol as a contributor to aberrant reward processing.

Expression of Key Modulators of Serotonergic Neurotransmission after Chronic Metformin Treatment

Despite metformin’s popularity as a treatment for metabolic and developmental disorders its mechanisms of action are still unknown. The goals of this project were to (1) assess the behavioral benefits of chronic metformin treatment alone for core autism symptoms, (2) assess if metformin acts on melanocortin 4 (MCR4) receptors in the brain to alter hormone levels in serum, and (3) to determine if metformin increases brain levels of peroxisome proliferator‐activated receptors α, δ, and γ (PPARs), which modulate insulin signaling throughout the body and increase in the liver with chronic metformin treatment. Since BTBR social behavior is enhanced by selective serotonin reuptake inhibitors (SSRIs) such as sertraline, and their chronic use reduces or otherwise alters receptor and transporter expression in the brain, we hypothesized that with chronic metformin treatment, expression of key regulators of serotonin neurotransmission such as the serotonin transporter (SERT) or organic cation transporters (OCTs) responsible for serotonin clearance would be reduced. Also, given prior evidence that chronic SSRI treatment increases serotonin levels in brain extracellular fluid, we hypothesized that expression of serotonin receptors such as 5‐HT2A among others would be reduced. This compensatory response occurs with chronic SSRI treatments. To explore these hypotheses BTBR T+ Itpr3tf/J (BTBR) and C57BL/6J (C57) mice were given chronic metformin treatment for six weeks, with behavior tests concluding in week 5, before euthanasia and blood and brain collection. With chronic metformin we found social interaction preference and social dominance were enhanced in male but not female BTBR mice. Blood serum was analyzed for hormonal changes, with C57 metformin treated mice seeing a reduction in corticosterone compared to controls. PPARδ was lower for metformin treated female C57 mice than control females, while PPARγ was found to be different between strain controls. Chronic metformin elevated blood glucose levels in female but not male mice. Male mice showed no changes in MCR 4 or SERT expression. Immunohistochemistry revealed an increase in OCT3 labeling in the brain of a BTBR male treated with metformin compared to a BTBR control male. The density of 5‐HT2A receptors was decreased in the anterior olfactory nucleus for BTBR mice following chronic metformin treatment. Overall, these findings indicate chronic metformin treatments that enhance social behaviors in male, but not female BTBR mice also have divergent effects on blood sugar among sexes. We did not find any evidence of chronic metformin treatment altering MCR4 or SERT but there was a decrease in 5‐HT2A expression. Effects on hormones were not present for BTBR mice but corticosterone was lower for C57 metformin treated males than controls, and metformin may increase OCT expression in BTBR males.

IL-1Ra deficiency accelerates intervertebral disc degeneration in C57BL6J mice.

The expression of Interleukin‐1ß (IL‐1ß) and its antagonist and Interleukin‐1 receptor antagonist (IL‐1Ra) are correlated with greater human intervertebral disc (IVD) degeneration, suggesting that elevated IL‐1β activity promotes disc degeneration. Many in vitro studies support such a mechanistic relationship, whereas few in vivo investigations have been reported. The present study tests the effect of increased IL‐1β activity on intervertebral disc in mice with an IL‐1Ra gene deletion.IL‐1Ra−/− mice and wild‐type (WT) C57Bl6J mice were examined at 3 and 12 months of age. Caudal IVD segments were evaluated for disc degeneration by histopathology, functional testing, and inflammatory gene expression relevant to IL‐1β pathways. To test differences in injury response, pinprick annular puncture was performed on IL‐1Ra−/− and WT mice and evaluated similarly.IL‐1Ra−/− IVDs had significantly worse histopathology at 3 months compared to WT controls, but not at 12 months. IL‐1Ra−/− IVDs exhibited significantly more viscous mechanical properties than WT IVDs. qPCR revealed downregulation of inflammatory genes at 3 and 12 months in IL‐1Ra−/− IVDs, with concomitant downregulation of anabolic and catabolic genes. Annular puncture yielded no appreciable differences between 2‐week and 6‐week post‐injured WT and IL1‐Ra−/− IVDs in histopathology or biomechanics, but inflammatory gene expression was sharply downregulated in IL‐1Ra−/− mice at 2 weeks, returning by 6 weeks post injury.In the present study, IL‐1Ra deletion resulted in increased IVD histopathology, inferior biomechanics, and transiently decreased pro‐inflammatory cytokine gene expression. The histopathology of IL‐1Ra−/− IVDs on a C57BL/6J background is less severe than a previous report of IL1Ra−/− on a BALB/c background, yet both strains exhibit IVD degeneration, reinforcing a mechanistic role of IL‐1β signaling in IVD pathobiology. Despite a pro‐inflammatory environment, the annular puncture was no worse in IL‐1Ra−/− mice, suggesting that response to injury involves pathways other than inflammation. Overall, this study supports the hypothesis that IL‐1β‐driven inflammation is important in IVD degeneration.

Cowpea Mosaic Virus Outperforms Other Members of the Secoviridae as In Situ Vaccine for Cancer Immunotherapy

In situ vaccination for cancer immunotherapy uses intratumoral administration of small molecules, proteins, nanoparticles, or viruses that activate pathogen recognition receptors (PRRs) to reprogram the tumor microenvironment and prime systemic antitumor immunity. Cowpea mosaic virus (CPMV) is a plant virus that─while noninfectious toward mammals─activates mammalian PRRs. Application of CPMV as in situ vaccine (ISV) results in a potent and durable efficacy in tumor mouse models and canine patients; data indicate that CPMV outperforms small molecule PRR agonists and other nonrelated plant viruses and virus-like particles (VLPs). In this work, we set out to compare the potency of CPMV versus other plant viruses from the Secoviridae. We developed protocols to produce and isolate cowpea severe mosaic virus (CPSMV) and tobacco ring spot virus (TRSV) from plants. CPSMV, like CPMV, is a comovirus with genome and protein homology, while TRSV lacks homology and is from the genus nepovirus. When applied as ISV in a mouse model of dermal melanoma (using B16F10 cells and C57Bl6J mice), CPMV outperformed CPSMV and TRSV─again highlighting the unique potency of CPMV. Mechanistically, the increased potency is related to increased signaling through toll-like receptors (TLRs)─in particular, CPMV signals through TLR2, 4, and 7. Using knockout (KO) mouse models, we demonstrate here that all three plant viruses signal through the adaptor molecule MyD88─with CPSMV and TRSV predominantly activating TLR2 and 4. CPMV induced significantly more interferon β (IFNβ) compared to TRSV and CPSMV; therefore, IFNβ released upon signaling through TLR7 may be a differentiator for the observed potency of CPMV-ISV. Additionally, CPMV induced a different temporal pattern of intratumoral cytokine generation characterized by significantly increased inflammatory cytokines 4 days after the second of 2 weekly treatments, as if CPMV induced a "memory response". This higher, longer-lasting induction of cytokines may be another key differentiator that explains the unique potency of CPMV-ISV.

Activin A causes endothelial dysfunction of mouse aorta and human aortic cells.

Preeclampsia is a multisystem hypertensive disorder of pregnancy that remains one of the leading causes of maternal and perinatal morbidity and mortality worldwide. The widespread maternal endothelial dysfunction that underlies preeclampsia is thought to arise from excessive placental production of various factors combined with enhanced oxidative stress. While previous studies have reported elevated activin A in women diagnosed with preeclampsia, whether activin A can cause vascular dysfunction has not yet been thoroughly investigated. Here, we demonstrated that different subtypes of activin A receptors were localised to the endothelial and smooth muscle cells of mouse and human aortae. Then, the aorta of healthy female C57Bl6J mice (n = 8) were incubated for 24 h in various concentrations of recombinant activin A to mimic early pregnancy (5 ng/mL), late pregnancy (20 ng/mL) and preeclampsia (50 ng/mL). Vascular reactivity as assessed by wire myography revealed that only the preeclamptic level of activin A impaired agonist-mediated endothelium-dependent relaxation by reducing the vasodilator prostanoid contribution to relaxation. However, agonist-mediated endothelium-independent mechanisms were unaffected. Further investigations carried out on human aortic endothelial cells suggested that the impairment of aorta relaxation could also be driven by increased endothelial cell permeability, and decreased cell viability, adherence and proliferation. This is the first direct evidence to show that activin A can induce endothelial dysfunction in whole blood vessels, suggesting that at high circulating levels it may contribute to the widespread endothelial dysfunction in women with preeclampsia.

Chinese Tea Alleviates CCl4-Induced Liver Injury through the NF-κBorNrf2Signaling Pathway in C57BL-6J Mice.

Liver injury is a life-threatening condition that is usually caused by excessive alcohol consumption, improperdiet, and stressful lifestyle and can even progress to liver cancer. Tea is a popular beverage with proven health benefits and is known to exert a protective effect on the liver, intestines, and stomach. In this study, we analyzed the therapeutic effects of six kinds of tea on carbon tetrachloride (CCl4)-induced liver injury in a mouse model. The mice were injected with 10 mL/kg 5% CCl4 to induce liver injury and then given oral gavage of green tea, yellow tea, oolong tea, white tea, black tea, and dark tea, respectively. The serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured, and the expression levels of inflammation and oxidative stress-related proteins in the liver tissues were quantified. All six kinds of tea partly reduced the liver index, restored the size of the enlarged liver in the CCl4 model, and decreased the serum levels of ALT and AST. Furthermore, the highly fermented dark tea significantly reduced the expression levels of NF-κB and the downstream inflammatory factors, whereas the unfermented green tea inhibited oxidative stress by activating the antioxidant Nrf2 pathway. Taken together, tea can protect against liver inflammation, and unfermented tea can improve antioxidant levels. Further studies are needed on the bioactive components of tea to develop drugs against liver injury.