Alzet Osmotic Minipumps Research Articles

The Impact of Hydrogen Sulfide in the Paraventricular Nucleus on the MAPK Pathway in High Salt-Induced Hypertension.

The hypothalamic paraventricular nucleus (PVN) plays a central role in regulating cardiovascular activity and blood pressure. We administered hydroxylamine hydrochloride (HA), a cystathionine-β-synthase inhibitor, into the PVN to suppress endogenous hydrogen sulfide and investigate its effects on the mitogen-activated protein kinase (MAPK) pathway in high salt (HS)-induced hypertension. We randomly divided 40 male Dahl salt-sensitive rats into 4 groups: the normal salt (NS) + PVN vehicle group, the NS + PVN HA group, the HS + PVN vehicle group, and the HS + PVN HA group, with 10 rats in each group. The rats in the NS groups were fed a NS diet containing 0.3% NaCl, while the HS groups were fed a HS diet containing 8% NaCl. The mean arterial pressure was calculated after noninvasive measurement using an automatic sphygmomanometer to occlude the tail cuff once a week. HA or vehicle was infused into the bilateral PVN using Alzet osmotic mini pumps for 6 weeks after the hypertension model was successfully established. We measured the levels of H 2 S in the PVN and plasma norepinephrine using enzyme linked immunosorbent assay. In addition, we assessed the parameters of the MAPK pathway, inflammation, and oxidative stress through western blotting, immunohistochemical analysis, or real-time polymerase chain reaction. In this study, we discovered that decreased levels of endogenous hydrogen sulfide in the PVN contributed to the onset of HS-induced hypertension. This was linked to the activation of the MAPK signaling pathway, proinflammatory cytokines, and oxidative stress in the PVN, as well as the activation of the sympathetic nervous system.

CD147 monoclonal antibody attenuates abdominal aortic aneurysm formation in angiotensin II-Infused apoE-/- mice.

Abdominal aortic aneurysm (AAA) is a life threatening vascular disease. Our previous study reported the upregulation of CD147 expression in human aortic aneurysms. In this study, we injected apoE-/- mice intraperitoneally with CD147 monoclonal antibody or IgG control antibody to observe its effect on Angiotensin II (AngII) induced AAA formation. ApoE-/- mice were randomly divided into an AngⅡ+CD147 antibody group (n=20) and an AngⅡ+IgG antibody group (n=20). The Alzet osmotic minipump was implanted subcutaneously into the backs of mice to infuse AngII (1000ng/kg/min) for 28days and subsequently treated with CD147 monoclonal antibody or control IgG mAb (10μg/mouse/day) beginning one day after surgery. Body weight, food intake, drinking volume and blood pressure were measured weekly throughout the study. After 4weeks of injection, routine bloodwork measuring liver function, kidney function and lipid levels were recorded. Hematoxylin and eosin (H&E), Masson's trichrome, and Elastic van Gieson (EVG) staining were used to evaluate the pathological changes in blood vessels. In addition, Immunohistochemical assay was used to detect infiltration of inflammatory cells. Tandem mass tag (TMT)-based proteomic analysis was used to define differentially expressed proteins (DEPs) using a p-value<0.05 and fold change>1.2 or<0.83 as the threshold. Subsequently, we conducted protein-protein interaction (PPI) network and GO enrichment analysis to determine the core biological function altered after CD147 antibody injection. The CD147 monoclonal antibody suppresses Ang II-induced AAA formation in apoE-/- mice and reduced aortic expansion, elastic lamina degradation, and inflammatory cells accumulation. Bioinformatics analysis showed that Ptk6, Itch, Casp3, and Oas1a were the hub DEPs. These DEPs in the two group were mainly involved in collagen fibril organization, extracellular matrix organization, and muscle contraction. These data robustly demonstrated that CD147 monoclonal antibody suppresses Ang II-induced AAA formation through reduction of inflammatory response and regulation of the above defined hub proteins and biological processes. Thus, the CD147 monoclonal antibody might be a promising target in the treatment of abdominal aortic aneurysm.

Effects of Na/K‐ATPase Inhibitor Marinobufagenin on Gene Expression and Cognition in a Mouse Model of Alzheimer’s Disease

AbstractBackgroundAlzheimer’s disease (AD) is characterized by brain amyloid‐β plaque formation, neuroinflammation and neurodegeneration, which lead to cognitive impairment (CI) and disruption of circadian rhythm. Bioactive steroidal Na/K‐ATPase inhibitor marinobufagenin (MBG) modulates neuroinflammation. In 16‐mo old double transgenic APPswe/PS1dE9 AD mice with advanced AD, treatment with MBG reduced mRNA expression of inflammatory markers. Here, we investigated whether treatment with MBG at early‐stage AD may impact AD manifestation in this AD mouse model.MethodsFive months old male AD mice (n = 15) and WT mice (n = 28) were administered MBG (100 µg/day/kg body weight) (AD‐MBG, n = 8; WT‐MBG, n = 14) or vehicle for control (AD‐C, n = 7; WT‐C, n = 14) via subcutaneous ALZET osmotic minipumps for 3 months. At 8‐mo of age, the mice underwent turn‐based discrimination learning in a water T‐maze (WTM) to assess procedural learning and 72‐hour home cage activity (HCA) analysis to assess circadian rhythm. The hippocampal inflammatory and AD mRNAs (by qPCR) and MBG levels (by immunoassay) were measured.ResultsMBG administration increased plasma MBG levels in WT and AD mice (Table 1). AD‐C mice exhibited greater home cage activity disruption vs. WT‐C (Fig.1a); in WTM, AD‐C required more trials to learn the turn‐based task than WT‐C (Fig. 1b). Though statistically insignificant, trends showed that MBG numerically worsened activity disruption by HCA and decreased ability to learn turn‐based task by WTM in WT mice, while numerically improving both measures in AD mice. During the reversal phase of WTM where response requirements were reversed, WT‐C mice performed significantly better than AD‐C mice, measured by the cumulative amount of time spent in the incorrect arm until reaching criterion performance (p = 0.038). Similar patterns of numerical improvement in AD‐MBG compared to AD‐C are shown (Fig. 1c). Hippocampal inflammatory and AD markers mRNAs were upregulated in AD‐C vs. WT‐C, and MBG treatment downregulated the expression of TNF and GFAP in AD mice (Table 1).ConclusionsBehavioral testing results illustrate CI and circadian impairment in 8‐mo old AD mice compared to WT. Numerical trends show a potential protective effect of MBG on AD CI likely via downregulation of hippocampal TNF and GFAP genes.0Supported by the NIH/NIA Intramural Research Program

PS-BPB06-6: RENAL INJURY ASSOCIATED WITH ARGININE VASOPRESSIN INDUCTION IN A RAT MODEL OF PREECLAMPSIA

Objective: Worldwide, preeclampsia (PE) accounts for 2–8% of pregnancies and is a main cause of maternal and fetal morbidity and mortality. In vivo murine studies confirm the use of arginine vasopressin (AVP) in replicating the clinical symptoms of PE development, suggestive of a role for PE prediction. The renal effects that AVP may have in PE remains elusive, hence this study aimed to explore the physiological effects of AVP on the kidneys of pregnant rats in an effort to determine its role in PE development. Design and method: Twenty-four female Sprague Dawley rats (non-pregnant saline, pregnant saline, non-pregnant AVP and pregnant AVP) were surgically implanted with ALZET mini osmotic pumps infusing with either saline or AVP for 18 days. Blood pressure measurements and urine samples were taken on gestational days (GD) 8, 14 and 18. Placental and fetal weights were recorded following sacrifice on GD18. Urinary levels of kidney injury molecule-1 (KIM-1) were determined using the rat kidney toxicity immunoassay panel 1 (#RKTX1MAG-37K). Immunohistochemistry was used to determine the expression of KIM-1 in rat kidney tissue. Results: Systolic [143.00 (139.67–144.00) mmHg vs 125.00 (124.00–127.00) mmHg; p &lt; 0.05] and diastolic blood pressures [103.50 (100.33–106.88) mmHg vs 81.00 (80.00–87.00) mmHg; p &lt; 0.001] were significantly higher in the pregnant AVP vs pregnant saline group. Urinary protein levels in pregnant AVP rats [0.45 (0.42–0.48) g/L] were significantly elevated in comparison to pregnant saline rats [0.24 (0.21–0.32) g/L; p &lt; 0.05]. Pregnant AVP rats also demonstrated a significant decrease in individual placental [0.39 (0.31–0.46) g vs 0.59 (0.55–0.63) g; p &lt; 0.05] and pup weights [1.57 (1.45–1.67) g vs 1.79 (1.70–1.85) g; p &lt; 0.05] in comparison to pregnant saline rats. Urinary KIM-1 levels are significantly elevated in the pregnant AVP (0.53 ± 0.05 ng/mL) vs pregnant saline group (0.17 ± 0.02 ng/mL; p &lt; 0.001). Qualitative analysis revealed that KIM-1 expression is markedly upregulated on the apical membrane surface of epithelial cells of the proximal convoluted tubules in AVP treated rats. Conclusions: AVP infusion increases blood pressure, urine protein levels and urinary and tissue expression of KIM-1. Our findings indicate that there is significant kidney injury associated with AVP treatment, indicative of the utility of this model in studying the mechanisms driving renal damage in PE.

Ca2+/Calmodulin-Dependent Protein Kinase II Regulation by Inhibitor of RIPK3 Protects against Cardiac Hypertrophy.

The activity of Ca2+/calmodulin-dependent protein kinase II δ (CaMKII δ) is central to the mechanisms of cardiovascular diseases. Receptor-interacting protein kinase 3- (RIPK3-) mediated necroptosis has been reported to contribute to cardiac dysfunction. However, the potential protective role of inhibition of RIPK3, a regulator of CaMKII, on cardiac hypertrophy remains unclear. The present study is aimed at investigating how the RIPK3 inhibitor GSK'872 regulates CaMKII activity and exploring its effect on hypertrophic cardiomyopathy (HCM). Wild-type (WT) and RIPK3 gene knockout (RIPK3−/−) mice were implanted subcutaneously with Alzet miniosmotic pumps (200 μL) and perfused with angiotensin II (AMP-AngII) to induce cardiac hypertrophy. After WT mice were induced by AngII for 72 hours, they were injected with GSK'872 with an intraperitoneal (IP) dose of 6 mg/kg once a day for two weeks. After this, they were physiologically examined for Echocardiography, myocardial injury, CaMKII activity, necroptosis, RIPK3 expression, mixed lineage kinase domain-like protein (MLKL) phosphorylation, and mitochondrial ultrastructure. The results indicated that deletion of the RIPK3 gene or administration of GSK'872 could reduce CaMKII activity, alleviate oxidative stress, reduce necroptosis, and reverse myocardial injury and cardiac dysfunction caused by AngII-induced cardiac hypertrophy in mice. The present study demonstrated that CaMKII activation and necroptosis augment cardiac hypertrophy in a RIPK3-dependent manner, which may provide therapeutic strategies for HCM. RIPK3 inhibitor GSK'872 has a protective effect on cardiac hypertrophy and could be an efficacious targeted medicine for HCM in clinical treatment.

P-474 Development of a preclinical screening pipeline to test the efficacy of anti-fibrotic and anti-inflammatory drugs on ovarian aging

Abstract Study question Can either systemic or targeted ovarian delivery of anti-inflammatory (Etanercept) or anti-fibrotic (Pirfenidone) drugs have a therapeutic effect on ovarian aging in mice? Summary answer We have established an in vitro and in vivo pre-clinical pipeline in the mouse to evaluate the effect of drugs on ovarian aging. What is known already The female reproductive system is the first to age in humans, beginning when women reach their mid-thirties. Reproductive aging results in infertility and hormonal perturbations which have general health consequences. Most research on reproductive aging has focused on the age-associated reduction in oocyte quantity and quality. However, our lab discovered that the ovarian microenvironment (stroma) in which the gamete develops changes with age. Specifically, a pro-inflammatory milieu develops with a significant increase in pro-inflammatory cytokine production including interleukin-6. Concurrently, collagen I/III accumulates resulting in fibrosis. This fibrotic and inflammatory milieu likely impacts oocyte quality contributing to reproductive aging. Study design, size, duration Etanercept was selected as an anti-inflammatory drug because it binds and inhibits TNF-a/b. Pirfenidone was selected as anti-fibrotic drug because it targets collagen I/III and TGF-b1. In vitro studies were performed using tissue explant-cultures to evaluate the toxicity of the drugs on the ovary. In vivo studies were based on the delivery of these drugs either systemically or targeted to the ovary using mini-osmotic pumps to evaluate the impact of these drugs on ovarian aging. Participants/materials, setting, methods In vitro studies: Ovaries from 7-month (N = 6) and 14-month (N = 6) old CD1 mice were isolated, dissected into equal pieces, and incubated in Pirfenidone (0.05, 0.5, 5mM), Etanercept (1, 10, 100g/ml), or the drug diluent (controls). After 24h the explants were collected and tissue architecture (H&amp;E), collagen content (Picrosirius red/PSR), and cell death (Caspase-3) was analyzed using histochemical approaches. In vivo studies: Alzet mini-osmotic pumps were implanted subcutaneously and 2-delivery approaches tested: systemic (N = 2) and ovarian-targeted(N = 2). Main results and the role of chance We first evaluated the toxicity of Etanercept and Pirfenidone on the ovary using our in vitro system. H&amp;E staining revealed that the structure of the Etanercept and Pirfenidone-treated ovaries were similar to controls. The collagen content was also not significantly different in all groups (p &amp;gt; 0.05, One-way ANOVA, Tukey’s test). However, 5mM Pirfenidone and 10 and 100g/ml Etanercept treatments resulted in a trend of decreased collagen relative to the control ovaries. Cleaved Caspase-3 fluorescent intensity/area was also similar in all conditions (p &amp;gt; 0.05, One-way ANOVA, Tukey’s test). Therefore, our in vitro studies show that Pirfenidone and Etanercept are not toxic demonstrating the potential use of these drugs in the ovary. Next, we tested the use of these drugs in vivo with osmotic pumps in 7-month old mice, when ovarian fibrosis is first observed. As a proof-of-concept, the pump was filled with Alcian Blue (dye). Pumps were implanted subcutaneously for systemic and targeted delivery. For the targeted delivery, the pump was connected to a catheter which was inserted underneath the bursa. The pumps were well tolerated by the mice. Two weeks after surgery the catheters were still attached to the ovaries and none of the ovaries showed structural abnormalities when analyzed with H&amp;E. Limitations, reasons for caution We have established a preclinical pipeline to screen the potential of drugs to extend reproductive longevity. Studies are ongoing to determine the efficacy of Pirfenidone and Etanercept in delaying fibrosis and inflammation in the ovary to improve reproductive outcomes. Experiments to determine the side effects of these drugs are required. Wider implications of the findings Our in vitro studies show that Pirfenidone and Etanercept are not toxic to the ovary suggesting a therapeutic potential of anti-fibrotic and anti-inflammatory drugs in delaying reproductive aging. Our in vivo preclinical pipeline represents a novel system to evaluate the therapeutic effect of these and other drugs on ovarian aging. Trial registration number N/A

Impact of synemin protein, an intermediate filament, in cardiac physiopathology

Synemin is a protein belonging to the intermediate filament family involved in the cytoarchitecture of skeletal, cardiac and smooth muscle cells. Synemin copolymerizes with other intermediate filaments (desmin, vimentin) and interacts with sarcomeric and costameric proteins, acting as bridging protein. The tail domain of synemin ties the type II α regulatory (R) subunit of protein kinase A (PKA). Therefore, synemin is an A-kinase-anchoring protein (AKAP) that allows and modulates spatial and temporal targeting of its activity. We have previously found that mechanical overload of skeletal muscle induces greater hypertrophy of muscle fibres in synemin-knockout mice associated with an impairment of PKA signalling pathways. In humans, two missense mutations, located near the AKAP domain, have been recorded in patients with dilated cardiomyopathy. The aims are: (1) To analyze the role of synemin in cardiac hypertrophy; (2) To characterize the signalling pathways related to synemin in the hypertrophic response. Morphological (body and heart weights), cellular (immunostaining) and molecular (RT-qPCR, Western blot) analyzes were carried out on cardiomyocyte-specific synemin-knockout mice at 10, 30 and 60 days after tamoxifen injections. Then, we studied the response of the hypertrophic agent angiotensin II (Ang II) after 14 days delivered by Alzet osmotic mini-pumps in these mice. Our data show a decrease (> 50%) of synemin expression in heart of conditional knockout mice 10 days after tamoxifen injections. Thus, we observed an increase of stress gene expression (ANF, βMHC) and fibrotic markers (vimentin, coll3α1). Immunofluorescence of vimentin on heart sections reveals a strong increase of protein expression suggesting an activation of fibroblast proliferation. Chronic administration of Ang II does not show modification in gene expression profile between control and conditional synemin-knockout mice. At 30 and 60 days after tamoxifen injections, we observed the maintenance of mRNA synemin level indicating a compensatory expression of this protein by other cells in heart (fibroblasts, macrophages, endothelial and smooth cells). Our data indicate a crucial role of synemin as modulator in the physiopathological conditions. Moreover, the relevance of synemin-PKA coupling in cardiac hypertrophy will be characterized in the near future.

Bioactive Steroid Marinobufagenin in a Mouse Model of Early‐Stage Alzheimer's Disease

BackgroundAlzheimer’s disease (AD) is characterized by brain amyloid beta plaque formation, neuroinflammation and neuronal degradation, which lead to cognitive impairment and decline in normal circadian rhythm (CR). Bioactive steroid marinobufagenin (MBG) modulates neuroinflammation. In 16‐mo old double transgenic APPswe/PS1dE9 AD mice with advanced AD, amyloid precursor protein (APP) and interleukin 6 (IL6) mRNAs were upregulated vs. wild type (WT) control, and treatment with MBG reduced mRNA expression. Here, we investigated whether treatment with MBG at early‐stage AD may impact AD development in this AD mouse model.MethodsFive months old male AD mice and WT mice were administered MBG (100 µg/day/kg) (AD‐MBG, n= 8; WT‐MBG, n= 14) or vehicle for control (AD‐C, n= 7; WT‐C, n= 14) via ALZET osmotic minipumps for 3 months. At 8‐mo of age, the mice underwent turn‐based discrimination learning in a water T‐maze (WTM) to assess procedural learning and home cage activity (HCA) analysis during three consecutive days of single housing. Higher number of trials required to achieve criterion indicates greater difficulty with learning the task in WTM. The inflammatory and AD gene profile (by qPCR) and MBG levels (by immunoassay) were measured in hippocampus and plasma. Data was analyzed by 2‐way ANOVA followed by Tukey’s test and presented as mean ±SE.ResultsWT‐C and AD‐C mice had similar plasma MBG levels (243 ± 34 vs. 307 ± 65 pmol/L). MBG was higher in WT‐MBG (447 ± 59 pmol/L; p &lt; 0.05 vs. WT‐C) and AD‐MBG mice (554 ± 57 pmol/L; p &lt; 0.05 vs. AD‐C). In HCA, AD‐C showed greater disruption in nightly CR, represented by greater activation during the first 6 hours of the night (Fig. 1a, b). While MBG did not significantly affect behavioral performance in WT or AD mice, trends show that MBG may worsen CR in WT and improve CR in AD mice. In WTM, AD‐C required more trials than WT‐C to reach criterion (Fig. 1c). MBG treatment numerically decreased the number of trials in AD‐MBG vs. AD‐C. MBG shows potential protective effect against cognitive impairment in AD mice. WT‐MBG exhibited numerical increase in the number of trials, thus, MBG may reduce procedural learning ability in WT mice in WTM (Fig. 1c). Hippocampal inflammatory marker IL10 mRNA was 2‐fold lower (p&lt;0.01) and IL6 mRNA was the same in AD‐C vs. WT‐C. AD markers were higher in AD‐C vs. WT‐C: APP mRNA 1.7‐fold (P&lt;0.01) and glial fibrillary acidic protein (GFAP) mRNA 1.6‐fold (p&lt;0.05). MBG treatment did not affect this gene expression.ConclusionsAD mice in comparison to WT mice exhibited early‐stage AD procedural learning impairment and CR disorder, which were accompanied by upregulated hippocampal AD markers without inflammatory marker activation. MBG treatment may have a protective effect against early‐stage AD‐related learning impairment and CR disruption. Future studies will examine the effects of MBG on the different cognitive domains in AD mice.

Treatment of Experimental Autoimmune Encephalomyelitis with an Inhibitor of Phosphodiesterase-8 (PDE8).

After decades of development, inhibitors targeting cyclic nucleotide phosphodiesterases (PDEs) expressed in leukocytes have entered clinical practice for the treatment of inflammatory disorders, with three PDE4 inhibitors being in clinical use as therapeutics for psoriasis, psoriatic arthritis, chronic obstructive pulmonary disease and atopic dermatitis. In contrast, the PDE8 family that is upregulated in pro-inflammatory T cells is a largely unexplored therapeutic target. We have previously demonstrated a role for the PDE8A-Raf-1 kinase complex in the regulation of myelin oligodendrocyte glycoprotein peptide 35–55 (MOG35–55) activated CD4+ effector T cell adhesion and locomotion by a mechanism that differs from PDE4 activity. In this study, we explored the in vivo treatment of experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis (MS) induced in mice immunized with MOG using the PDE8-selective inhibitor PF-04957325. For treatment in vivo, mice with EAE were either subcutaneously (s.c.) injected three times daily (10 mg/kg/dose), or were implanted subcutaneously with Alzet mini-osmotic pumps to deliver the PDE8 inhibitor (15.5 mg/kg/day). The mice were scored daily for clinical signs of paresis and paralysis which were characteristic of EAE. We observed the suppression of the clinical signs of EAE and a reduction of inflammatory lesion formation in the CNS by histopathological analysis through the determination of the numbers of mononuclear cells isolated from the spinal cord of mice with EAE. The PDE8 inhibitor treatment reduces the accumulation of both encephalitogenic Th1 and Th17 T cells in the CNS. Our study demonstrates the efficacy of targeting PDE8 as a treatment of autoimmune inflammation in vivo by reducing the inflammatory lesion load.

Effect of Hydro-Alcoholic Root Extract of Delphinium Denudatum Wall on Mecamylamine-Precipitated Nicotine-Withdrawal and Corticosterone Levels in Rats

This study evaluates the effects of hydro-alcoholic root extract of Delphinium denudatum Wall (Jadwar, family; Ranunculaceae) for its ability to attenuate the nicotine-withdrawal in nicotine-dependent rats. The physical dependence on nicotine was induced in male adult Wistar albino rats (175-250 g) by subcutaneous implantation of Alzet mini osmotic pumps which supplied nicotine at 9.0mg/kg/day, while control rats received saline via osmotic pumps. For seven days four separate test doses of hydro-alcoholic root extract of Delphinium denudatum Wall (200, 400, 800, 1600 mg/kg) were given orally. On the 7th day, a mecamylamine injection (1 mg/kg, s.c.) was given to precipitate nicotine-withdrawal. The global Gellert’s–Holtzman rating scale was used to rate somatic signs of withdrawal for 15 minutes, followed by a measurement of motor activity. Drug Bupropion was used as a positive control. Serum levels of nicotine (cotinine) and corticosterone were done by ELISA. In nicotine-dependent rats, oral administration of Delphinium denudatum Wall root extract suppressed the hyper-locomotion and decreased the corticosterone levels at all dosages. Higher doses (800 and 1600 mg/kg) of extract, significantly attenuated nicotine withdrawal whereas, lower doses (200 and 400 mg/kg) had no significant effect. These results suggest that hydro-alcoholic root extract of Delphinium denudatum Wall may prove to be a potential therapeutic agent to attenuate nicotine dependence and facilitate tobacco smoking cessation.

Retraction Note to: Treatment with a brain-selective prodrug of 17β-estradiol improves cognitive function in Alzheimer’s disease mice by regulating klf5-NF-κB pathway

10β,17β-dihydroxyestra-1,4-dien-3-one (DHED) which is a brain-selective prodrug of 17β-estradiol has been reported to improve the cognitive function in Alzheimer’s disease (AD) mice model. However, little is known about the potential mechanism for cognitive improvement. In the present study, we used AD mice to investigate the effects and mechanisms of DHED treatment. Female Tg2576 transgenic AD mice were ovariectomized and then treated by implanting Alzet osmotic minipumps containing DHED or vehicle subcutaneously for 8 weeks. Consistent with previous report, DHED treatment ameliorated cognitive function of AD mice with decreasing Aβ levels in the hippocampus. Besides, we also found DHED treatment could reduce oxidative and inflammatory stress and the level of p-tau. The mechanisms underlying the cognitive function improvement may be linked with estrogen receptor (ER)-klf5-NF-κB pathway, demonstrated by decreased expression of klf5 and the secretion of inflammatory cytokines. However, the effects of DHED treatment could be reversed when ERα was inhibited by ICI182780. Taken together, our findings uncovered a new mechanism for DHED to improve the cognitive function of AD mice and may provide a viable therapy to treat AD.

Luteolin attenuates angiotensin II‑induced renal damage in apolipoprotein E‑deficient mice.

Renal damage is a common and severe condition encountered in the clinic. Luteolin (Lut) exhibits anti-inflammatory, anti-fibrotic and anti-apoptotic effects. Thus, the present study aimed to investigate the pharmacological effects of Lut on angiotensin II (AngII)-induced renal damage in apolipoprotein E-deficient (ApoE−/−) mice. Male ApoE−/− mice (age, 8 weeks) were randomly divided into the following three groups: i) Control group (n=6); ii) AngII group (n=6); and iii) AngII + Lut group (n=6). Lut was administered by gavage (100 mg/kg/d). ApoE−/− mice were implanted with Alzet osmotic minipumps, filled with either saline vehicle or AngII solution for a maximum period of 4 weeks. After 4 weeks, metabolic characteristics were measured and the histopathological alterations in the kidney tissue were observed. The metabolic characteristics of blood creatinine (CRE) levels were lower in the AngII + Lut group compared with in the AngII group. The expression levels of collagen I and III were lower in the kidney tissues of the AngII + Lut group compared with the corresponding tissues of the AngII group. The gene expression levels of IL-1β, IL-6, TNF-α and IL-10 were also suppressed in the kidney tissues of the AngII + Lut group compared with those in the corresponding tissues of the AngII group. Furthermore, the AngII + Lut group exhibited markedly increased LC3 protein expression and notably decreased p62 protein expression in the kidney tissues compared with the expression levels in the AngII group. The data demonstrated that Lut attenuated AngII-induced collagen deposition and inflammation, while inducing autophagy. Collectively, the results suggested that Lut treatment exhibited a exerted effect on AngII-induced renal injury in ApoE−/− mice.

Steroidal Inhibitors of Na/K‐ATPase in a Mouse Model of Alzheimer’s Disease

BackgroundNeurons rely on Na/K‐ATPase (NKA) to create the electrochemical gradient that allows them to fire action potentials. The NKA also acts as a receptor for cardiotonic steroids (CTS), which have been shown to participate in intracellular signaling transduction, cardiovascular function, and have potential neuroprotective effects. Despite the importance of the NKA and CTS in brain physiology and pathology, their potential roles in Alzheimer’s disease (AD) pathology are not well studied. Marinobufagenin (MBG) is a bufadienolide CTS, and its potential neuroprotective effect has never been studied. MBG levels were significantly lower in 15‐month old double‐mutant Alzheimer’s mice (APPswe/PS1dE9; 2xTg‐AD; AD) compared to age‐matched, non‐transgenic controls. The present study examined whether the treatment of AD mice by MBG affects cardiovascular and behavioral parameters.MethodsMale AD (n=16) and wild type (WT; n=8) mice (15‐mo old) were included in the experimental design. Mice were administered MBG (100 μg/day/kg body weight; AD‐MBG, n=8; WT‐MBG, n=4) or vehicle (control: AD‐C, n=8; WT‐C, n=4) treatment using subcutaneous ALZET osmotic minipumps over the course of 12 weeks before data (Systolic blood pressure (SBP; by tail cuff plethysmography), open field test behavior (OFT), and plasma, tissues samples) was collected. Data was analyzed by 2‐way ANOVA and t‐test and presented as mean±SEM.ResultsSBP did not differ between WT‐C and AD‐C mice. AD mice weighed more than WT (P&lt;0.05). MBG did not affect either parameters (Table 1). WT‐MBG mice had higher heart rates than WT‐C mice (Table 1, P&lt;0.05). Plasma MBG was lower in AD‐C mice vs. WT‐C (125±21 pmol/L vs. 223±41 pmol/L, P&lt;0.05, t‐test). MBG increased plasma levels of this steroid in both WT‐MBG and AD‐MBG mice, which indicated the efficiency of the drug delivery (Table 1). AD‐C mice had 1.6‐fold higher brain cortical expression of inflammatory marker interleukin‐6 (IL6) mRNA vs. WT‐C; MBG significantly reduced cortical mRNA IL6 expression in AD‐MBG mice (Fig 1). No differences were found for total distance traveled, nor for time spent in the center during first 90 seconds of OFT between AD‐C and WT‐C, and between AD‐MBG and AD‐C. Distance traveled in the center showed an effect of strain (P&lt;0.05), an effect of treatment (P&lt;0.05), and a trend‐level treatment:strain interaction (P&lt;0.1). WT traveled farther in the center of the field vs. AD mice. MBG treated mice traveled less in the center vs. non‐treated mice. MBG caused a greater reduction in distance traveled in the center for WT mice than it did for AD mice (Fig 1).ConclusionAD mice had lower endogenous plasma MBG and higher brain pro‐inflammatory mRNA marker IL6 vs. WT mice. Treatment with MBG significantly reduced levels of this inflammatory marker. WT mice exhibited more exploratory behavior than AD mice in the OFT; whereas, MBG decreased exploratory behavior. Exploratory behavior in WT‐MBG mice was more affected than AD‐MBG mice. The association of the brain inflammatory response, cognitive functions and MBG will be further explored in this AD mouse model.Support or Funding InformationSupported by the NIH/NIA Intramural Research ProgramEffect of MBG treatment on expression of IL6 mRNA and OFT performance during first 90 seconds of task. IL6 mRNA (a), time spent in center (b), distance traveled in center (c), and total distance traveled (d) in wild type (WT) and 2xTg‐AD (AD) mice. By unpaired T‐test (a) †P&lt;0.05, WT vs. AD, #P&lt;0.05 AD‐C vs AD‐MBG; by 2‐way ANOVA Linear Mixed Effects followed by Tukey post‐test (b–d): *P&lt;0.05 WT vs. AD; ##P&lt;0.05 Vehicle vs. MBG: drug x strain interaction: P=0.06.Figure 1 Clinical, physiological and behavioral parameters in 15 months old AD mice and the wild type control mice with and without 12‐week treatment with MBG WT AD Control (n=4) MBG (n=4) Control (n=8) MBG (n=8) Body weight, g 35.0 ± 1.3 34.5 ± 1.8 38.1 ± 1.6 * 38.1 ± 0.5 SBP, mm Hg 98.2 ± 4.3 100.0 ± 4.3 91.6 ± 1.0 103.6 ± 5.9 HR, beats/min 457 ± 11 594 ± 4 513 ± 29 557 ± 19 # Plasma MBG, pmol/L 223 ± 41 483 ± 116 125 ± 21 † 657 ± 176 # OFT distance center, m 0.44 ± 0.14 0.11 ± 0.04 0.17 ± 0.04 * 0.15 ± 0.03 # OFT distance total, m 3.66 ± 0.48 2.56 ± 0.47 2.37 ± 0.48 2.79 ± 1.09 OFT time center, s 4.48 ± 1.84 2.48 ± 1.44 3.01 ± 1.38 0.92 ± 0.38 Data presented as Mean ± SEM. By 2‐way ANOVA followed by Tukey post‐test: *P&lt;0.05, AD vs. WT; #P&lt;0.05. MBG vs. control. By unpaired t‐test: †P&lt;0.05. AD vs. WT. For OFT distance center, effect of strain: P=0.03: effect of drug, P=0.02; drug x strain interaction: P=0.06. Post‐hoc: WT‐MBG HR higher than WT‐C HR (P&lt;0.05). WT, wild type mice: AD, 2xTg‐AD mice; MBG, marinobufagenin; SBP, systolic blood pressure; HR, heart rate: OFT, openfield test.

Chronic Staphylococcus aureus Superantigen Toxic Shock Syndrome Toxin-1 Exposure Accelerates the Progression of Atherosclerosis in Rabbits.

It has been reported that infectious agents contribute to the atherosclerotic process. However, it is unclear whether Staphylococcus aureus superantigen (SAg) toxic shock syndrome toxin-1 (TSST-1) has an effect on atherosclerosis progression. The present study was designed to investigate the pathogenic role of TSST-1 exposure in the atherosclerotic process in rabbits. New Zealand White rabbits were exposed to TSST-1 through Alzet miniosmotic pumps with a constant pumping rate. Aortic atherosclerosis was evaluated by histological and morphometric methods. Using a biochemical analyzer/enzyme-linked immunosorbent assay/immunostaining, we further analyzed various atherosclerosis-related factors. The gross atherosclerotic lesion area in the aortic arch increased by 15.3% in high-fat-diet rabbits exposed to TSST-1 compared to that in the control group. In the atherosclerotic lesions, TSST-1 exposure increased the content of smooth muscle cells. Moreover, TSST-1 treatment up-regulated serum tumor necrosis factor alpha (TNF-α) level, but did not affect serum lipids (except for triglycerides) and endotoxin in the rabbits. Our data validated that chronic stimulation with TSST-1 facilitates the progression of atherosclerosis in rabbits independently of endotoxins, indicating that S. aureus and its SAgs may be targets to prevent and treat atherosclerosis.

Morphine immunomodulation prolongs inflammatory and postoperative pain while the novel analgesic ZH853 accelerates recovery and protects against latent sensitization

BackgroundNumerous studies have identified the proinflammatory, pronociceptive effects of morphine which ultimately exacerbate pain. Our novel endomorphin analog ZH853 does not produce proinflammatory effects on its own and gives potent, long-lasting analgesia. This study investigates whether ZH853’s lack of interaction with the neuroimmune system reduces the risk of prolonged pain.MethodsAdult male Sprague-Dawley rats were subjected to one of two treatment paradigms. Either (1) chronic pain followed by chronic treatment with morphine, ZH853 or vehicle, or (2) chronic drug administered prior to pain induction. Complete Freund’s adjuvant (CFA) was injected or paw incision surgery was performed on the left hind plantar foot pad. Drugs were administered through Alzet osmotic minipumps at a rate of 1 μl/h for 5 days at appropriate doses based on prior experiments. Animals were tested for mechanical allodynia and thermal hyperalgesia using von Frey filaments and the Hargreaves apparatus, respectively. Additionally, several gait parameters were measured using the CatWalk XT. When all animals had recovered from pain, 1 mg/kg of naltrexone was administered to test for development of latent sensitization (LS). A second set of animals was used to investigate dorsal horn inflammation following CFA and drug treatment. ANOVAs were used to assess differences between drug treatment groups.ResultsAs expected, morphine increased and prolonged pain in all experiments compared to vehicle treatment. However, ZH853 treatment reduced the overall time spent in pain and the severity of pain scores compared to morphine. ZH853 not only reduced inflammation versus morphine treatment but also, in some instances, acted as an anti-inflammatory drug compared to vehicle treatment. Finally, ZH853 prevented the development of LS while vehicle- and morphine-treated animals showed robust relapse to pain.ConclusionsZH853 has a favorable side effect profile versus morphine and provides superior analgesia in a number of pain states. We now know that chronic use of this compound reduces time spent in a chronic pain state, the opposite of common opioids like morphine, and reduces the risk of LS, making ZH853 an excellent candidate for clinical development in humans for inflammatory and postoperative pain.

Biomarkers and smart intracranial devices for the diagnosis, treatment, and monitoring of high-grade gliomas: a review of the literature and future prospects.

Glioblastoma (GBM) is the most common primary brain neoplasm with median overall survival (OS) around 15 months. There is a dearth of effective monitoring strategies for patients with high-grade gliomas. Relying on magnetic resonance images of brain has its challenges, and repeated brain biopsies add significant morbidity. Hence, it is imperative to establish a less invasive way to diagnose, monitor, and guide management of patients with high-grade gliomas. Currently, multiple biomarkers are in various phases of development and include tissue, serum, cerebrospinal fluid (CSF), and imaging biomarkers. Here we review and summarize the potential biomarkers found in blood and CSF, including extracellular macromolecules, extracellular vesicles, circulating tumor cells, immune cells, endothelial cells, and endothelial progenitor cells. The ability to detect tumor-specific biomarkers in blood and CSF will potentially not only reduce the need for repeated brain biopsies but also provide valuable information about the heterogeneity of tumor, response to current treatment, and identify disease resistance. This review also details the status and potential scope of brain tumor-related cranial devices and implants including Ommaya reservoir, microelectromechanical systems-based depot device, Alzet mini-osmotic pump, Metronomic Biofeedback Pump (MBP), ipsum G1 implant, ultra-thin needle implant, and putative devices. An ideal smart cranial implant will overcome the blood-brain barrier, deliver various drugs, provide access to brain tissue, and potentially measure and monitor levels of various biomarkers.

Adrenergic modulation of AMPK‑dependent autophagy by chronic stress enhances cell proliferation and survival in gastric cancer.

Epidemiological data show that chronic stress has adverse effects on the incidence and progression of cancer. As a critical target organ for stress hormones, the stomach is frequently subjected to stress-related injury. However, few reports regarding the association between stress and gastric cancer (GC) have been published. The present study aimed to investigate the effect of chronic stress on the growth and survival of GC, and the role of the autophagy process. A restraint-stress procedure over 21 days was used to establish a chronic stress mouse model. Subcutaneous xenografts and gastric orthotopic xenografts were established in BALB/c nude mice. Alzet osmotic minipumps containing either PBS or propranolol hydrochloride was inserted on the nape of the neck 7 days prior to the initiation of restraint stress. The presence of autophagosomes and autolysosomes were examined by electron microscopy. The stress hormone norepinephrine significantly enhanced the proliferation of GC cells. By inhibiting adrenoreceptor expression, it was demonstrated that β2-adrenergic receptor (ADRB2) was the specific β-adrenergic receptor subtype responsible for catecholamine release. In addition, it was demonstrated that the induction of autophagy was a novel consequence of β2-adrenergic activation in GC cells. This was demonstrated by the appearance of double-membrane vesicles, punctuate GFP-RFP-microtubule-associated protein 1 light chain 3 distribution in the cytoplasm and a corresponding increase in autophagic flux. Notably, norepinephrine-induced autophagy was shown to have a tumor-promoting role under conditions of chronic stress in vitro and in vivo. It was further demonstrated that, upon activation of cAMP-response element binding protein, chronic stress promoted autophagic flux through the adenosine 5′-monophosphate-activated protein kinase-unc-51 like autophagy activating kinase 1 (AMPK-ULK1) pathway. Tissue microarray analysis revealed a negative correlation between the expression of ADRB2 and autophagic marker p62/sequestosome-1 in GC tumor samples. Additionally, high protein levels of ADRB2 correlated positively with tumor, node, metastasis stage and poor prognosis in patients with GC. These results establish a novel pathway that chronic stress activates tumor-promoting autophagy to accelerate the progression of GC. The present study is the first, to the best of our knowledge, providing preclinical evidence that chronic stress serves a role in the progression of GC.

RETRACTED ARTICLE: Treatment with a brain-selective prodrug of 17\u03b2-estradiol improves cognitive function in Alzheimer\u2019s disease mice by regulating klf5-NF-\u03baB pathway

10β,17β-dihydroxyestra-1,4-dien-3-one (DHED) which is a brain-selective prodrug of 17β-estradiol has been reported to improve the cognitive function in Alzheimer’s disease (AD) mice model. However, little is known about the potential mechanism for cognitive improvement. In the present study, we used AD mice to investigate the effects and mechanisms of DHED treatment. Female Tg2576 transgenic AD mice were ovariectomized and then treated by implanting Alzet osmotic minipumps containing DHED or vehicle subcutaneously for 8 weeks. Consistent with previous report, DHED treatment ameliorated cognitive function of AD mice with decreasing Aβ levels in the hippocampus. Besides, we also found DHED treatment could reduce oxidative and inflammatory stress and the level of p-tau. The mechanisms underlying the cognitive function improvement may be linked with estrogen receptor (ER)-klf5-NF-κB pathway, demonstrated by decreased expression of klf5 and the secretion of inflammatory cytokines. However, the effects of DHED treatment could be reversed when ERα was inhibited by ICI182780. Taken together, our findings uncovered a new mechanism for DHED to improve the cognitive function of AD mice and may provide a viable therapy to treat AD.

NFAT5 regulates renal gene expression in response to angiotensin II through Annexin-A2-mediated posttranscriptional regulation in hypertensive rats

The aim was to identify new targets that regulate gene expression at the posttranscriptional level in angiotensin II (ANGII)-mediated hypertension. Heparin affinity chromatography was used to enrich nucleic acid-binding proteins from kidneys of two-kidney, one-clip (2K1C) hypertensive Wistar rats. The experiment was repeated with 14-day ANGII infusion using Alzet osmotic mini pumps, with or without ANGII receptor AT1a inhibition using losartan in the drinking water. Mean arterial pressure increased after 2K1C or ANGII infusion and was inhibited with losartan. Heparin affinity chromatography and mass spectrometry were used to identify Annexin-A2 (ANXA2) as having differential nucleic acid-binding activity. Total Annexin-A2 protein expression was unchanged, whereas nucleic acid-binding activity was increased in both kidneys of 2K1C and after ANGII infusion through AT1a stimulation. Costaining of Annexin-A2 with α-smooth muscle actin and aquaporin 2 showed prominent expression in the endothelia of larger arteries and the cells of the inner medullary collecting duct. The nuclear factor of activated T cells (NFAT) transcription factor was identified as a likely Annexin-A2 target using enrichment analysis on a 2K1C microarray data set and identifying several binding sites in the regulatory region of the mRNA. Expression analysis showed that ANGII increases NFAT5 protein but not mRNA level and, thus, indicated that NFAT5 is regulated by posttranscriptional regulation, which correlates with activation of the RNA-binding protein Annexin-A2. In conclusion, we show that ANGII increases Annexin-A2 nucleic acid-binding activity that correlates with elevated protein levels of the NFAT5 transcription factor. NFAT signaling appears to be a major contributor to renal gene regulation in high-renin states.

Abstract 108: Lipopolysaccharide Fails to Augment Development of Angiotensin II-induced Abdominal Aortic Aneurysms in Mice

Background and objective: Our previous study demonstrated that deficiency of toll-like receptor 4 (TLR4) reduced angiotensin II (AngII)-induced abdominal aortic aneurysms (AAAs) in hypercholesterolemic mice. Lipopolysaccharide (LPS) is a well-known agonist of TLR4. Interaction of LPS and TLR4 promotes inflammation and augments hypercholesterolemia-induced atherosclerosis. The purpose of this study was to determine whether LPS augments AngII-induced AAAs. Methods and Results: Low-density lipoprotein (LDL) receptor -/- mice fed a Western diet develop hypercholesterolemia and have augmentation of AngII-induced AAAs. We found that LDL receptor -/- mice fed Western diet had increased LPS concentrations, as quantified by a Limulus Amebocyte lysate chromogenic endotoxin quantitation kit, compared to the same mice prior to Western diet feeding. To determine whether LPS plays a role on AngII-induced AAAs, 4 groups of male C57BL/6J mice, 10-week-old, were infused subcutaneously with: (1) Vehicle (N=5), (2) LPS (250 μ g/kg/day; N=10), (3) AngII (1,000 ng/kg/min; N=10), or (4) both AngII and LPS (N=10) through Alzet mini osmotic pumps for 28 days. Abdominal aortic expansion was monitored serially at 2 and 4 weeks of infusion using high frequency ultrasonography (Vevo 3100 system; FUJIFILM). Continuous luminal expansion was detected in both AngII-infused mice and mice co-infused with both AngII and LPS. These results were confirmed by ex vivo measurements of maximal outer diameters of suprarenal aortas after termination showing AngII infusion led to significant increase of abdominal aortic dilation, compared to vehicle or LPS infused mice. However, co-infusion of LPS with AngII did not augment AngII-induced AAAs. Conclusion: LPS has no effects on AngII-induced AAAs in normolipidemic mice.