Cross-Fostering Increases Th1/Th2 Expression in a Prenatal Dexamethasone Exposure Rat Model

Background

Prenatal dexamethasone exposure has been reported to increase allergy potential in childhood possibly by interference with normal immunological development in utero. This study investigated the effects of prenatal dexamethasone on T helper cell immune responses in a rat model.

Methods

Pregnant rats received either dexamethasone 0.1 mg/kg/day or normal saline from gestational day 14–21. Off-springs were cared for by their biological mother, or cross-fostered by the opposing group. Spleen and blood samples were collected at post-natal day 7 and 120 and tested for mRNA expression and plasma cytokine levels of Th1/Th2/Th17 immune response.

Results

Both Th1 (T-bet) and Th2 (GATA-3) mRNA expression were shown to have a significant increase in the prenatal dexamethasone exposure group at day 120 (p<0.05). The plasma levels for Th1 (IFNγ and IL-2) and Th2 (IL-4, IL-5, IL-13) were found to have no significant differences between the two group (p>0.05). The mRNA expression of Th17 (RORγt) showed a significant decrease at post-natal day 120 as well as the plasma level of IL-17A at day 7 (11.21±1.67 vs. 6.23±1.06 pg/ml, p = 0.02). Cross-fostering by a dexamethasone exposed mother resulted in a significant increase in Th1/Th2 mRNA expression (p<0.05) and decrease of Th17.

Conclusions

Prenatal dexamethasone exposure increased Th1, Th2 and decreased Th17 expression. Cross-fostering by a dexamethasone exposed mother results in more prominent increase of Th1 and Th2 expression.     

Ag85B DNA vaccine suppresses airway inflammation in a murine model of asthma

Background

In allergic asthma, Th2 lymphocytes are believed to play important roles in orchestrating airway eosinophilia and inflammation. Resetting the Th1/Th2 imbalance may have a therapeutic role in asthma. The mycobacterium tuberculosis 30-kilodalton major secretory protein (antigen 85B, Ag85B) can protect animals from M. tuberculosis infection by inducing a Th1-dominant response.

Methods

In this study, the Ag85B gene was cloned into pMG plasmids to yield the pMG-Ag85B plasmid. The expression of Ag85B gene in murine bronchial epithelia cells was detected by Western blotting and immunohistochemical staining after intranasal immunization with reconstructed pMG-Ag85B plasmids. The protective effect of pMG-Ag85B plasmids immunization in airway inflammation was evaluated by histological examination and bronchoalveolar lavage (BAL). IL-4 and IFN-γ levels in the BAL and supernatant from splenocyte culture were determined using ELISA kits.

Results

The Ag85B gene was successfully expressed in murine bronchial epithelia cells by intranasal immunization with reconstructed pMG-Ag85B plasmids. Using a murine model of asthma induced by ovalbumin (OVA), pMG-Ag85B immunization significantly inhibited cellular infiltration across the airway epithelium with a 37% decrease in the total number of cells (9.6 ± 2.6 × 10⁵/ml vs. 15.2 ± 3.0 × 10⁵/ml, p < 0.05) and a 74% decrease in the number of eosinophils (1.4 ± 0.2 × 10⁵/ml vs. 5.4 ± 1.1 × 10⁵/ml, p < 0.01) compared with the OVA-sensitized control group. There was no difference in the number of neutrophils in BAL fluid between the pMG-Ag85B group, the OVA-sensitized control group and the empty pMG group. IL-4 production was significantly decreased in the BAL fluid (32.0 ± 7.6 pg/ml vs. 130.8 ± 32.6 pg/ml, p < 0.01) and in the splenocyte supernatant (5.1 ± 1.6 pg/ml vs. 10.1 ± 2.3 pg/ml, p < 0.05) in the pMG-Ag85B group compared with the OVA-sensitized control group, while IFN-γ production was increased in the BAL fluid (137.9 ± 25.6 pg/ml vs. 68.4 ± 15.3 pg/ml, p < 0.05) and in the splenocyte supernatant (20.1 ± 5.4 pg/ml vs. 11.3 ± 3.2 pg/ml, p < 0.05).

Conclusion

In a murine model of asthma induced by OVA, intranasal immunization with pMG-Ag85B significantly reduced allergic airway inflammation with less eosinophil infiltration. This protective effect was associated with decreased IL-4 and increased IFN-γ production in the BAL fluid and in the supernatant of cultured splenocytes.     

High-Intensity Endurance Training Results in Faster Vessel-Specific Rate of Vasorelaxation in Type 1 Diabetic Rats

This study examined the effects of 6 weeks of moderate- (MD) and high-intensity endurance training (HD) and resistance training (RD) on the vasorelaxation responsiveness of the aorta, iliac, and femoral vessels in type 1 diabetic (D) rats. Vasorelaxation to acetylcholine was modeled as a mono-exponential function. A potential mediator of vasorelaxation, endothelial nitric oxide synthase (e-NOS) was determined by Western blots. Vessel lumen-to-wall ratios were calculated from H&E stains. The vasorelaxation time-constant (τ) (s) was smaller in control (C) (7.2±3.7) compared to D (9.1±4.4) and it was smaller in HD (5.4±1.5) compared to C, D, RD (8.3±3.7) and MD (8.7±3.8) (p<0.05). The rate of vasorelaxation (%·s⁻¹) was larger in HD (2.7±1.2) compared to C (2.0±1.2), D (2.0±1.5), RD (2.0±1.0), and MD (2.0±1.2) (p<0.05). τ vasorelaxation was smaller in the femoral (6.9±3.7) and iliac (6.9±4.7) than the aorta (9.0±5.0) (p<0.05). The rate of vasorelaxation was progressively larger from the femoral (3.1±1.4) to the iliac (2.0±0.9) and to the aorta (1.3±0.5) (p<0.05). e-NOS content (% of positive control) was greater in HD (104±90) compared to C (71±64), D (85±65), RD (69±43), and MD (76±44) (p<0.05). e-NOS normalized to lumen-to-wall ratio (%·mm⁻¹) was larger in the femoral (11.7±11.1) compared to the aorta (3.2±1.9) (p<0.05). Although vasorelaxation responses were vessel-specific, high-intensity endurance training was the most effective exercise modality in restoring the diabetes-related loss of vascular responsiveness. Changes in the vasoresponsiveness seem to be endothelium-dependent as evidenced by the greater e-NOS content in HD and the greater normalized e-NOS content in the smaller vessels.     

Comparison of Retinal Nerve Fiber Layer Thickness In Vivo and Axonal Transport after Chronic Intraocular Pressure Elevation in Young versus Older Rats

Purpose

To compare in young and old rats longitudinal measurements of retinal nerve fiber layer thickness (RNFLT) and axonal transport 3-weeks after chronic IOP elevation.

Method

IOP was elevated unilaterally in 2- and 9.5-month-old Brown-Norway rats by intracameral injections of magnetic microbeads. RNFLT was measured by spectral domain optical coherence tomography. Anterograde axonal transport was assessed from confocal scanning laser ophthalmolscopy of superior colliculi (SC) after bilateral intravitreal injections of cholera toxin-B-488. Optic nerve sections were graded for damage.

Results

Mean IOP was elevated in both groups (young 37, old 38 mmHg, p = 0.95). RNFL in young rats exhibited 10% thickening at 1-week (50.9±8.1 µm, p<0.05) vs. baseline (46.4±2.4 µm), then 7% thinning at 2-weeks (43.0±7.2 µm, p>0.05) and 3-weeks (43.5±4.4 µm, p>0.05), representing 20% loss of dynamic range. RNFLT in old rats showed no significant change at 1-week (44.9±4.1 µm) vs. baseline (49.2±5.3 µm), but progression to 22% thinning at 2-weeks (38.0±3.7 µm, p<0.01) and 3-weeks (40.0±6.6 µm, p<0.05), representing 59% loss of dynamic range. Relative SC fluorescence intensity was reduced in both groups (p<0.001), representing 77–80% loss of dynamic range and a severe transport deficit. Optic nerves showed 75–95% damage (p<0.001). There was greater RNFL thinning in old rats (p<0.05), despite equivalent IOP insult, transport deficit and nerve damage between age groups (all p>0.05).

Conclusion

Chronic IOP elevation resulted in severely disrupted axonal transport and optic nerve axon damage in all rats, associated with mild RNFL loss in young rats but a moderate RNFL loss in old rats despite the similar IOP insult. Hence, the glaucomatous injury response within the RNFL depends on age.     

Phenolic Acid Composition,Antiatherogenic and Anticancer Potential of Honeys Derived from Various Regions in Greece

The phenolic acid profile of honey depends greatly on its botanical and geographical origin. In this study, we carried out a quantitative analysis of phenolic acids in the ethyl acetate extract of 12 honeys collected from various regions in Greece. Our findings indicate that protocatechuic acid, p-hydroxybenzoic acid, vanillic acid, caffeic acid and p-coumaric acid are the major phenolic acids of the honeys examined. Conifer tree honey (from pine and fir) contained significantly higher concentrations of protocatechuic and caffeic acid (mean: 6640 and 397 µg/kg honey respectively) than thyme and citrus honey (mean of protocatechuic and caffeic acid: 437.6 and 116 µg/kg honey respectively). p-Hydroxybenzoic acid was the dominant compound in thyme honeys (mean: 1252.5 µg/kg honey). We further examined the antioxidant potential (ORAC assay) of the extracts, their ability to influence viability of prostate cancer (PC-3) and breast cancer (MCF-7) cells as well as their lowering effect on TNF- α-induced adhesion molecule expression in endothelial cells (HAEC). ORAC values of Greek honeys ranged from 415 to 2129 µmol Trolox equivalent/kg honey and correlated significantly with their content in protocatechuic acid (p<0.001), p-hydroxybenzoic acid (p<0.01), vanillic acid (p<0.05), caffeic acid (p<0.01), p-coumaric acid (p<0.001) and their total phenolic content (p<0.001). Honey extracts reduced significantly the viability of PC-3 and MCF-7 cells as well as the expression of adhesion molecules in HAEC. Importantly, vanillic acid content correlated significantly with anticancer activity in PC-3 and MCF-7 cells (p<0.01, p<0.05 respectively). Protocatechuic acid, vanillic acid and total phenolic content correlated significantly with the inhibition of VCAM-1 expression (p<0.05, p<0.05 and p<0.01 respectively). In conclusion, Greek honeys are rich in phenolic acids, in particular protocatechuic and p-hydroxybenzoic acid and exhibit significant antioxidant, anticancer and antiatherogenic activities which may be attributed, at least in part, to their phenolic acid content.     

Notch-1 Mediated Cardiac Protection following Embryonic and Induced Pluripotent Stem Cell Transplantation in Doxorubicin-Induced Heart Failure

Doxorubicin (DOX), an effective chemotherapeutic drug used in the treatment of various cancers, is limited in its clinical applications due to cardiotoxicity. Recent studies suggest that transplanted adult stem cells inhibit DOX-induced cardiotoxicity. However, the effects of transplanted embryonic stem (ES) and induced pluripotent stem (iPS) cells are completely unknown in DOX-induced left ventricular dysfunction following myocardial infarction (MI). In brief, C57BL/6 mice were divided into five groups: Sham, DOX-MI, DOX-MI+cell culture (CC) media, DOX-MI+ES cells, and DOX-MI+iPS cells. Mice were injected with cumulative dose of 12 mg/kg of DOX and 2 weeks later, MI was induced by coronary artery ligation. Following ligation, 5×10⁴ ES or iPS cells were delivered into the peri-infarct region. At day 14 post-MI, echocardiography was performed, mice were sacrificed, and hearts were harvested for further analyses. Our data reveal apoptosis was significantly inhibited in ES and iPS cell transplanted hearts compared with respective controls (DOX-MI+ES: 0.48±0.06% and DOX-MI+iPS: 0.33±0.05% vs. DOX-MI: 1.04±0.07% and DOX-MI+CC: 0.96±0.21%; p<0.05). Furthermore, a significant increase in levels of Notch-1 (p<0.05), Hes1 (p<0.05), and pAkt (p<0.05) were observed whereas a decrease in the levels of PTEN (p<0.05), a negative regulator of Akt, was evident following stem cell transplantation. Moreover, hearts transplanted with stem cells demonstrated decreased vascular and interstitial fibrosis (p<0.05) as well as MMP-9 expression (p<0.01) compared with controls. Additionally, heart function was significantly improved (p<0.05) in both cell-transplanted groups. In conclusion, our data show that transplantation of ES and iPS cells blunt DOX-induced adverse cardiac remodeling, which is associated with improved cardiac function, and these effects are mediated by the Notch pathway.     

Increased Circulating ANG II and TNF-α Represents Important Risk Factors in Obese Saudi Adults with Hypertension Irrespective of Diabetic Status and BMI

Central adiposity is a significant determinant of obesity-related hypertension risk, which may arise due to the pathogenic inflammatory nature of the abdominal fat depot. However, the influence of pro-inflammatory adipokines on blood pressure in the obese hypertensive phenotype has not been well established in Saudi subjects. As such, our study investigated whether inflammatory factors may represent useful biomarkers to delineate hypertension risk in a Saudi cohort with and without hypertension and/or diabetes mellitus type 2 (DMT2). Subjects were subdivided into four groups: healthy lean controls (age: 47.9±5.1 yr; BMI: 22.9±2.1 Kg/m²), non-hypertensive obese (age: 46.1±5.0 yr; BMI: 33.7±4.2 Kg/m²), hypertensive obese (age: 48.6±6.1 yr; BMI: 36.5±7.7 Kg/m²) and hypertensive obese with DMT2 (age: 50.8±6.0 yr; BMI: 35.3±6.7 Kg/m²). Anthropometric data were collected from all subjects and fasting blood samples were utilized for biochemical analysis. Serum angiotensin II (ANG II) levels were elevated in hypertensive obese (p<0.05) and hypertensive obese with DMT2 (p<0.001) compared with normotensive controls. Systolic blood pressure was positively associated with BMI (p<0.001), glucose (p<0.001), insulin (p<0.05), HOMA-IR (p<0.001), leptin (p<0.01), TNF-α (p<0.001) and ANG II (p<0.05). Associations between ANG II and TNF-α with systolic blood pressure remained significant after controlling for BMI. Additionally CRP (p<0.05), leptin (p<0.001) and leptin/adiponectin ratio (p<0.001) were also significantly associated with the hypertension phenotype. In conclusion our data suggests that circulating pro-inflammatory adipokines, particularly ANG II and, TNF-α, represent important factors associated with a hypertension phenotype and may directly contribute to predicting and exacerbating hypertension risk.     

Complement Alternative Pathway Activation in Human Nonalcoholic Steatohepatitis

The innate immune system plays a major role in the pathogenesis of nonalcoholic steatohepatitis (NASH). Recently we reported complement activation in human NASH. However, it remained unclear whether the alternative pathway of complement, which amplifies C3 activation and which is frequently associated with pathological complement activation leading to disease, was involved. Here, alternative pathway components were investigated in liver biopsies of obese subjects with healthy livers (n = 10) or with NASH (n = 12) using quantitative PCR, Western blotting, and immunofluorescence staining. Properdin accumulated in areas where neutrophils surrounded steatotic hepatocytes, and colocalized with the C3 activation product C3c. C3 activation status as expressed by the C3c/native C3 ratio was 2.6-fold higher (p<0.01) in subjects with NASH despite reduced native C3 concentrations (0.94±0.12 vs. 0.57±0.09; p<0.01). Hepatic properdin levels positively correlated with levels of C3c (r_s = 0.69; p<0.05) and C3c/C3 activation ratio (r_s = 0.59; p<0.05). C3c, C3 activation status (C3c/C3 ratio) and properdin levels increased with higher lobular inflammation scores as determined according to the Kleiner classification (C3c: p<0.01, C3c/C3 ratio: p<0.05, properdin: p<0.05). Hepatic mRNA expression of factor B and factor D did not differ between subjects with healthy livers and subjects with NASH (factor B: 1.00±0.19 vs. 0.71±0.07, p = 0.26; factor D: 1.00±0.21 vs. 0.66±0.14, p = 0.29;). Hepatic mRNA and protein levels of Decay Accelerating Factor tended to be increased in subjects with NASH (mRNA: 1.00±0.14 vs. 2.37±0.72; p = 0.22; protein: 0.51±0.11 vs. 1.97±0.67; p = 0.28). In contrast, factor H mRNA was downregulated in patients with NASH (1.00±0.09 vs. 0.71±0.06; p<0.05) and a similar trend was observed with hepatic protein levels (1.12±0.16 vs. 0.78±0.07; p = 0.08). Collectively, these data suggest a role for alternative pathway activation in driving hepatic inflammation in NASH. Therefore, alternative pathway factors may be considered attractive targets for treating NASH by inhibiting complement activation.     

Change of Th17 Lymphocytes and Treg/Th17 in Typical and Atypical Optic Neuritis

Background

Typical and atypical optic neuritis (ON) are two clinical types of autoimmune inflammatory diseases of the optic nerve that causes acute vision loss, and are difficult to distinguish in their early stages. The disturbance in the balance of Th17 and Treg lymphocytes is thought to play an essential role in these autoimmune inflammatory diseases.

Objectives

To detect the clinical relevance of Th17 and Treg in peripheral blood and the ratio of Treg/Th17 in patients with typical and atypical ON. To determine whether analysis of Th17 and Treg lymphocytes will provides insights into the different disease phenotypes of typical and atypical ON.

Methods

We studied a consecutive series of patients aged 14–70 years who presented to our neurological department with typical ON (n = 30) or atypical ON (n = 33) within 4 weeks of their acute attacks. Routine clinical tests and ophthalmological examination were performed in all patients. Blood samples were collected from untreated patients and from gender- and age-matched healthy controls (n = 30). The proportion of peripheral blood Th17 cells and Treg cells was determined by flow cytometry.

Results

Patients with atypical ON had a higher proportion of Th17 cells than patients with typical ON (3.61±1.56 vs 2.55±1.74, P<0.01) or controls (1.45±0.86, P<0.01). The proportion of Th17 cells in patients with typical ON was also markedly higher than in controls (P<0.01). The mean percentage of Treg cells in atypical ON (6.31±2.11) and typical ON (6.80±2.00) were significantly lower when compared to controls (8.29±2.32, both P<0.01). No significant difference in Treg frequency was observed between typical ON and atypical ON (p>0.05).

Conclusions

The frequency of Th17 cells is higher in atypical ON than typical ON, and patients with atypical ON have a greater imbalance of pro-inflammatory and regulatory cells than patients with typical ON when compared with controls. These changes are indicative of distinct pathological mechanisms and may provide useful information to distinguish typical and atypical ON.     

BMP7 Gene Transfer via Gold Nanoparticles into Stroma Inhibits Corneal Fibrosis In Vivo

This study examined the effects of BMP7 gene transfer on corneal wound healing and fibrosis inhibition in vivo using a rabbit model. Corneal haze in rabbits was produced with the excimer laser performing -9 diopters photorefractive keratectomy. BMP7 gene was introduced into rabbit keratocytes by polyethylimine-conjugated gold nanoparticles (PEI2-GNPs) transfection solution single 5-minute topical application on the eye. Corneal haze and ocular health in live animals was gauged with stereo- and slit-lamp biomicroscopy. The levels of fibrosis [α-smooth muscle actin (αSMA), F-actin and fibronectin], immune reaction (CD11b and F4/80), keratocyte apoptosis (TUNEL), calcification (alizarin red, vonKossa and osteocalcin), and delivered-BMP7 gene expression in corneal tissues were quantified with immunofluorescence, western blotting and/or real-time PCR. Human corneal fibroblasts (HCF) and in vitro experiments were used to characterize the molecular mechanism mediating BMP7’s anti-fibrosis effects. PEI2-GNPs showed substantial BMP7 gene delivery into rabbit keratocytes in vivo (2×10⁴ gene copies/ug DNA). Localized BMP7 gene therapy showed a significant corneal haze decrease (1.68±0.31 compared to 3.2±0.43 in control corneas; p<0.05) in Fantes grading scale. Immunostaining and immunoblot analyses detected significantly reduced levels of αSMA (46±5% p<0.001) and fibronectin proteins (48±5% p<0.01). TUNEL, CD11b, and F4/80 assays revealed that BMP7 gene therapy is nonimmunogenic and nontoxic for the cornea. Furthermore, alizarin red, vonKossa and osteocalcin analyses revealed that localized PEI2-GNP-mediated BMP7 gene transfer in rabbit cornea does not cause calcification or osteoblast recruitment. Immunofluorescence of BMP7-transefected HCFs showed significantly increased pSmad-1/5/8 nuclear localization (>88%; p<0.0001), and immunoblotting of BMP7-transefected HCFs grown in the presence of TGFβ demonstrated significantly enhanced pSmad-1/5/8 (95%; p<0.001) and Smad6 (53%, p<0.001), and decreased αSMA (78%; p<0.001) protein levels. These results suggest that localized BMP7 gene delivery in rabbit cornea modulates wound healing and inhibits fibrosis in vivo by counter balancing TGFβ1-mediated profibrotic Smad signaling.     

Fibroblast Growth Factor-9 Enhances M2 Macrophage Differentiation and Attenuates Adverse Cardiac Remodeling in the Infarcted Diabetic Heart

Inflammation has been implicated as a perpetrator of diabetes and its associated complications. Monocytes, key mediators of inflammation, differentiate into pro-inflammatory M1 macrophages and anti-inflammatory M2 macrophages upon infiltration of damaged tissue. However, the inflammatory cell types, which propagate diabetes progression and consequential adverse disorders, remain unclear. The current study was undertaken to assess monocyte infiltration and the role of fibroblast growth factor-9 (FGF-9) on monocyte to macrophage differentiation and cardioprotection in the diabetic infarcted heart. Db/db diabetic mice were assigned to sham, myocardial infarction (MI), and MI+FGF-9 groups. MI was induced by permanent coronary artery ligation and animals were subjected to 2D transthoracic echocardiography two weeks post-surgery. Immunohistochemical and immunoassay results from heart samples collected suggest significantly increased infiltration of monocytes (Mean ± SEM; MI: 2.02% ± 0.23% vs. Sham 0.75% ± 0.07%; p<0.05) and associated pro-inflammatory cytokines (TNF-α, MCP-1, and IL-6), adverse cardiac remodeling (Mean ± SEM; MI: 33% ± 3.04% vs. Sham 2.2% ± 0.33%; p<0.05), and left ventricular dysfunction (Mean ± SEM; MI: 35.4% ± 1.25% vs. Sham 49.19% ± 1.07%; p<0.05) in the MI group. Importantly, treatment of diabetic infarcted myocardium with FGF-9 resulted in significantly decreased monocyte infiltration (Mean ± SEM; MI+FGF-9: 1.39% ± 0.1% vs. MI: 2.02% ± 0.23%; p<0.05), increased M2 macrophage differentiation (Mean ± SEM; MI+FGF-9: 4.82% ± 0.86% vs. MI: 0.85% ± 0.3%; p<0.05) and associated anti-inflammatory cytokines (IL-10 and IL-1RA), reduced adverse remodeling (Mean ± SEM; MI+FGF-9: 11.59% ± 1.2% vs. MI: 33% ± 3.04%; p<0.05), and improved cardiac function (Fractional shortening, Mean ± SEM; MI+FGF-9: 41.51% ± 1.68% vs. MI: 35.4% ± 1.25%; p<0.05). In conclusion, our data suggest FGF-9 possesses novel therapeutic potential in its ability to mediate monocyte to M2 differentiation and confer cardiac protection in the post-MI diabetic heart.     

Magnesium Sulfate Treatment Reverses Seizure Susceptibility and Decreases Neuroinflammation in a Rat Model of Severe Preeclampsia

Eclampsia, defined as unexplained seizure in a woman with preeclampsia, is a life-threatening complication of pregnancy with unclear etiology. Magnesium sulfate (MgSO₄) is the leading eclamptic seizure prophylactic, yet its mechanism of action remains unclear. Here, we hypothesized severe preeclampsia is a state of increased seizure susceptibility due to blood-brain barrier (BBB) disruption and neuroinflammation that lowers seizure threshold. Further, MgSO₄ decreases seizure susceptibility by protecting the BBB and preventing neuroinflammation. To model severe preeclampsia, placental ischemia (reduced uteroplacental perfusion pressure; RUPP) was combined with a high cholesterol diet (HC) to cause maternal endothelial dysfunction. RUPP+HC rats developed symptoms associated with severe preeclampsia, including hypertension, oxidative stress, endothelial dysfunction and fetal and placental growth restriction. Seizure threshold was determined by quantifying the amount of pentylenetetrazole (PTZ; mg/kg) required to elicit seizure in RUPP+HC±MgSO₄ and compared to normal pregnant controls (n = 6/group; gestational day 20). RUPP+HC rats were more sensitive to PTZ with seizure threshold being ∼65% lower vs. control (12.4±1.7 vs. 36.7±3.9 mg/kg PTZ; p<0.05) that was reversed by MgSO₄ (45.7±8.7 mg/kg PTZ; p<0.05 vs. RUPP+HC). BBB permeability to sodium fluorescein, measured in-vivo (n = 5–7/group), was increased in RUPP+HC vs. control rats, with more tracer passing into the brain (15.9±1.0 vs. 12.2±0.3 counts/gram ×1000; p<0.05) and was unaffected by MgSO₄ (15.6±1.0 counts/gram ×1000; p<0.05 vs. controls). In addition, RUPP+HC rats were in a state of neuroinflammation, indicated by 35±2% of microglia being active compared to 9±2% in normal pregnancy (p<0.01; n = 3–8/group). MgSO₄ treatment reversed neuroinflammation, reducing microglial activation to 6±2% (p<0.01 vs. RUPP+HC). Overall, RUPP+HC rats were in a state of augmented seizure susceptibility potentially due to increased BBB permeability and neuroinflammation. MgSO₄ treatment reversed this, increasing seizure threshold and decreasing neuroinflammation, without affecting BBB permeability. Thus, reducing neuroinflammation may be one mechanism by which MgSO₄ prevents eclampsia during severe preeclampsia.     

Low Density Lipoproteins Promote Unstable Calcium Handling Accompanied by Reduced SERCA2 and Connexin-40 Expression in Cardiomyocytes

The damaging effects of high plasma levels of cholesterol in the cardiovascular system are widely known, but little attention has been paid to direct effects on cardiomyocyte function. We therefore aimed at testing the hypothesis that Low Density Lipoprotein (LDL) cholesterol affects calcium dynamics and signal propagation in cultured atrial myocytes. For this purpose, mRNA and protein expression levels were determined by real time PCR and western blot analysis, respectively, and intracellular calcium was visualized in fluo-4 loaded atrial HL-1 myocyte cultures subjected to field stimulation. At low stimulation frequencies all cultures had uniform calcium transients at all tested LDL concentrations. However, 500 µg LDL/mL maximally reduced the calcium transient amplitude by 43% from 0.30±0.04 to 0.17±0.02 (p<0.05). Moreover, LDL-cholesterol dose-dependently increased the fraction of alternating and irregular beat-to-beat responses observed when the stimulation interval was shortened. This effect was linked to a concurrent reduction in SERCA2, RyR2, IP3RI and IP3RII mRNA levels. SERCA2 protein levels were also reduced by 43% at 200 µg LDL/mL (p<0.05) and SR calcium loading was reduced by 38±6% (p<0.001). By contrast, HDL-cholesterol had no significant effect on SERCA expression or SR calcium loading. LDL-cholesterol also slowed the conduction velocity of the calcium signal from 3.2+0.2 mm/s without LDL to 1.7±0.1 mm/s with 500 µg LDL/mL (p<0.05). This coincided with a reduction in Cx40 expression (by 44±3%; p<0.05 for mRNA and by 79±2%; p<0.05 for Cx40 protein at 200 µg/ml LDL) whereas the Cx-43 expression did not significantly change. In conclusion, LDL-cholesterol destabilizes calcium handling in cultured atrial myocytes subjected to rapid pacing by reducing SERCA2 and Cx40 expression and by slowing the conduction velocity of the calcium signal.     

Intravascular Ultrasound Observation of the Mechanism of No-Reflow Phenomenon in Acute Myocardial Infarction

Objective

To study the mechanism of the no-reflow phenomenon using coronary angiography (CAG) and intravascular ultrasound (IVUS).

Methods

A total of 120 patients with acute myocardial infarction (AMI) who successfully underwent indwelling intracoronary stent placement by percutaneous coronary intervention (PCI). All patients underwent pre- and post-PCI CAG and pre-IVUS. No-reflow was defined as post-PCI thrombolysis in myocardial infarction (TIMI) grade 0, 1, or 2 flow in the absence of mechanical obstruction. Normal reflow was defined as TIMI grade 3 flow. The pre-operation reference vascular area, minimal luminal cross-sectional area, plaque cross-sectional area, lesion length, plaque volume and plaque traits were measured by IVUS.

Results

The no-reflow group was observed in 14 cases (11.6%) and normal blood-flow group in 106 cases (89.4%) based on CAG results. There was no statistically significant difference in the patients’ medical history, reference vascular area (no-flow vs. normal-flow; 15.5 ± 3.2 vs. 16.2 ± 3.3, p> 0.05) and lesion length (21.9 ± 5.1 vs. 19.5 ± 4.8, p> 0.05) between the two groups. No-reflow patients had a longer symptom onset to reperfusion time compared to normal blood-flow group [(6.6 ± 3.1) h vs (4.3 ± 2.7) h; p< 0.05] and higher incidence of TIMI flow grade< 3 (71.4% vs 49.0%, p< 0.05). By IVUS examination, the no-reflow group had a significantly increased coronary plaque area and plaque volume compared to normal blood-flow group [(13.7 ± 3.0) mm² vs (10.2 ± 2.9) mm²; (285.4 ± 99.8) mm³ vs (189.7 ± 86.4) mm³; p< 0.01]. The presence of IVUS-detected soft plaque (57.1% vs. 24.0%, p< 0.01), eccentric plaque (64.2% vs. 33.7%, p< 0.05), plaque rupture (50.0% vs. 21.2%, p< 0.01), and thrombosis (42.8% vs. 15.3%) were significantly more common in no-reflow group.

Conclusion

There was no obvious relationship between the coronary risk factors and no-reflow phenomenon. The symptom onset to reperfusion time, TIMI flow grade before stent deployment, plaque area, soft plaques, eccentric plaques, plaque rupture and thrombosis may be risk factors for the no-reflow phenomenon after PCI.     

Three Minutes of All-Out Intermittent Exercise per Week Increases Skeletal Muscle Oxidative Capacity and Improves Cardiometabolic Health

We investigated whether a training protocol that involved 3 min of intense intermittent exercise per week — within a total training time commitment of 30 min including warm up and cool down — could increase skeletal muscle oxidative capacity and markers of health status. Overweight/obese but otherwise healthy men and women (n = 7 each; age  = 29±9 y; BMI  = 29.8±2.7 kg/m²) performed 18 training sessions over 6 wk on a cycle ergometer. Each session began with a 2 min warm-up at 50 W, followed by 3×20 s “all-out” sprints against 5.0% body mass (mean power output: ∼450–500 W) interspersed with 2 min of recovery at 50 W, followed by a 3 min cool-down at 50 W. Peak oxygen uptake increased by 12% after training (32.6±4.5 vs. 29.1±4.2 ml/kg/min) and resting mean arterial pressure decreased by 7% (78±10 vs. 83±10 mmHg), with no difference between groups (both p<0.01, main effects for time). Skeletal muscle biopsy samples obtained before and 72 h after training revealed increased maximal activity of citrate synthase and protein content of cytochrome oxidase 4 (p<0.01, main effect), while the maximal activity of β-hydroxy acyl CoA dehydrogenase increased in men only (p<0.05). Continuous glucose monitoring measured under standard dietary conditions before and 48–72 h following training revealed lower 24 h average blood glucose concentration in men following training (5.4±0.6 vs. 5.9±0.5 mmol/L, p<0.05), but not women (5.5±0.4 vs. 5.5±0.6 mmol/L). This was associated with a greater increase in GLUT4 protein content in men compared to women (138% vs. 23%, p<0.05). Short-term interval training using a 10 min protocol that involved only 1 min of hard exercise, 3x/wk, stimulated physiological changes linked to improved health in overweight adults. Despite the small sample size, potential sex-specific adaptations were apparent that warrant further investigation.     

Reduced Ventricular Arrhythmogeneity and Increased Electrical Complexity in Normal Exercised Rats

Background

The mechanisms whereby aerobic training reduces the occurrence of sudden cardiac death in humans are not clear. We test the hypothesis that exercise-induced increased resistance to ventricular tachycardia and fibrillation (VT/VF) involve an intrinsic remodeling in healthy hearts.

Methods and Results

Thirty rats were divided into a sedentary (CTRL, n = 16) and two exercise groups: short- (4 weeks, ST, n = 7) and long-term (8 weeks, LT, n = 7) trained groups. Following the exercise program hearts were isolated and studied in a Langendorff perfusion system. An S₁–S₂ pacing protocol was applied at the right ventricle to determine inducibility of VT/VF. Fast Fourier transforms were applied on ECG time-series. In-vivo measurements showed training-induced increase in aerobic capacity, heart-to-body weight ratio and a 50% low-to-high frequency ratio reduction in the heart rate variability (p<0.05). In isolated hearts the probability for VF decreased from 26.1±14.4 in CTRL to 13.9±14.1 and 6.7±8.5% in the ST and LT, respectively (p<0.05). Duration of VF also decreased from 19.0±5.7 in CTRL to 8.8±7.1 and 6.0±5.8 sec in ST and LT respectively (p<0.05). Moreover, the pacing current required for VF induction increased following exercise (2.9±1.7 vs. 5.4±2.1 and 8.5±0.9 mA, respectively; p<0.05). Frequency analysis of ECG revealed an exercise-induced VF transition from a narrow single peak spectrum at 17 Hz in CTRL to a broader range of peaks ranging between 8.8 and 22.5 Hz in the LT group (p<0.05).

Conclusion

Exercise in rats leads to reduced VF propensity associated with an intrinsic cardiac remodeling related to a broader spectral range and faster frequency components in the ECG.     

Is There an Association between Sleeping Patterns and Other Environmental Factors with Obesity and Blood Pressure in an Urban African Population?

Beyond changing dietary patterns, there is a paucity of data to fully explain the high prevalence of obesity and hypertension in urban African populations. The aim of this study was to determine whether other environmental factors (including sleep duration, smoking and physical activity) are related to body anthropometry and blood pressure (BP). Data were collected on 1311 subjects, attending two primary health care clinics in Soweto, South Africa. Questionnaires were used to obtain data on education, employment, exercise, smoking and sleep duration. Anthropometric and BP measurements were taken. Subjects comprised 862 women (mean age 41 ± 16 years and mean BMI 29.9 ± 9.2 kg/m²) and 449 men (38 ± 14 years and 24.8 ± 8.3 kg/m²). In females, ANOVA showed that former smokers had a higher BMI (p<0.001) than current smokers, while exposure to second hand smoking was associated with a lower BMI (p<0.001) in both genders. Regression analyses demonstrated that longer sleep duration was associated with a lower BMI (p<0.05) in older females only, and not in males, whilst in males napping during the day for > 30 minutes was related to a lower BMI (β = -0.04, p<0.01) and waist circumference (β = -0.03, p<0.001). Within males, napping for >30 minutes/day was related to lower systolic (β = -0.02, p<0.05) and lower diastolic BP (β = -0.02, p = 0.05). Longer night time sleep duration was associated with higher diastolic (β = 0.005, p<0.01) and systolic BP (β = 0.003, p<0.05) in females. No health benefits were noted for physical activity. These data suggest that environmental factors rarely collected in African populations are related, in gender-specific ways, to body anthropometry and blood pressure. Further research is required to fully elucidate these associations and how they might be translated into public health programs to combat high levels of obesity and hypertension.     

Correlation of Perfusion MRI and 18F-FDG PET Imaging Biomarkers for Monitoring Regorafenib Therapy in Experimental Colon Carcinomas with Immunohistochemical Validation

ObjectivesTo investigate a multimodal, multiparametric perfusion MRI / ¹⁸F-fluoro-deoxyglucose-(¹⁸F-FDG)-PET imaging protocol for monitoring regorafenib therapy effects on experimental colorectal adenocarcinomas in rats with immunohistochemical validation.ResultsRegorafenib significantly (p<0.01) suppressed PF (81.1±7.5 to 50.6±16.0 mL/100mL/min), PV (12.1±3.6 to 7.5±1.6%) and PS (13.6±3.2 to 7.9±2.3 mL/100mL/min) as well as TTB (3.4±0.6 to 1.9±1.1) between baseline and day 7. Immunohistochemistry revealed significantly (p<0.03) lower tumor microvascular density (CD-31, 7.0±2.4 vs. 16.1±5.9) and tumor cell proliferation (Ki-67, 434.0 ± 62.9 vs. 663.0 ± 98.3) in the therapy group. Perfusion MRI parameters ΔPF, ΔPV and ΔPS showed strong and significant (r = 0.67-0.78; p<0.01) correlations to the PET parameter ΔTTB and significant correlations (r = 0.57-0.67; p<0.03) to immunohistochemical Ki-67 as well as to CD-31-stainings (r = 0.49-0.55; p<0.05).ConclusionsA multimodal, multiparametric perfusion MRI/PET imaging protocol allowed for non-invasive monitoring of regorafenib therapy effects on experimental colorectal adenocarcinomas in vivo with significant correlations between perfusion MRI parameters and ¹⁸F-FDG-PET validated by immunohistochemistry.     

SDF1-A Facilitates Lin?/Sca1+ Cell Homing following Murine Experimental Cerebral Ischemia

Background

Hematopoietic stem cells mobilize to the peripheral circulation in response to stroke. However, the mechanism by which the brain initiates this mobilization is uncharacterized.

Methods

Animals underwent a murine intraluminal filament model of focal cerebral ischemia and the SDF1-A pathway was evaluated in a blinded manner via serum and brain SDF1-A level assessment, Lin−/Sca1+ cell mobilization quantification, and exogenous cell migration confirmation; all with or without SDF1-A blockade.

Results

Bone marrow demonstrated a significant increase in Lin−/Sca1+ cell counts at 24 hrs (272±60%; P<0.05 vs sham). Mobilization of Lin−/Sca1+ cells to blood was significantly elevated at 24 hrs (607±159%; P<0.05). Serum SDF1-A levels were significant at 24 hrs (Sham (103±14), 4 hrs (94±20%, p = NS) and 24 hrs (130±17; p<0.05)). Brain SDF1-A levels were significantly elevated at both 4 hrs and 24 hrs (113±7 pg/ml and 112±10 pg/ml, respectively; p<0.05 versus sham 76±11 pg/ml). Following administration of an SDF1-A antibody, Lin−/Sca1+ cells failed to mobilize to peripheral blood following stroke, despite continued up regulation in bone marrow (stroke bone marrow cell count: 536±65, blood cell count: 127±24; p<0.05 versus placebo). Exogenously administered Lin−/Sca1+ cells resulted in a significant reduction in infarct volume: 42±5% (stroke alone), versus 21±15% (Stroke+Lin−/Sca1+ cells), and administration of an SDF1-A antibody concomitant to exogenous administration of the Lin−/Sca1+ cells prevented this reduction. Following stroke, exogenously administered Lin−/Sca1+ FISH positive cells were significantly reduced when administered concomitant to an SDF1-A antibody as compared to without SDF1-A antibody (10±4 vs 0.7±1, p<0.05).

Conclusions

SDF1-A appears to play a critical role in modulating Lin−/Sca1+ cell migration to ischemic brain.     

Cross-Sectional Comparison of Small Animal [18F]-Florbetaben Amyloid-PET between Transgenic AD Mouse Models

We aimed to compare [¹⁸F]-florbetaben PET imaging in four transgenic mouse strains modelling Alzheimer’s disease (AD), with the main focus on APPswe/PS2 mice and C57Bl/6 mice serving as controls (WT). A consistent PET protocol (N = 82 PET scans) was used, with cortical standardized uptake value ratio (SUVR) relative to cerebellum as the endpoint. We correlated methoxy-X04 staining of β-amyloid with PET results, and undertook ex vivo autoradiography for further validation of a partial volume effect correction (PVEC) of PET data. The SUVR in APPswe/PS2 increased from 0.95±0.04 at five months (N = 5) and 1.04±0.03 (p<0.05) at eight months (N = 7) to 1.07±0.04 (p<0.005) at ten months (N = 6), 1.28±0.06 (p<0.001) at 16 months (N = 6) and 1.39±0.09 (p<0.001) at 19 months (N = 6). SUVR was 0.95±0.03 in WT mice of all ages (N = 22). In APPswe/PS1G384A mice, the SUVR was 0.93/0.98 at five months (N = 2) and 1.11 at 16 months (N = 1). In APPswe/PS1dE9 mice, the SUVR declined from 0.96/0.96 at 12 months (N = 2) to 0.91/0.92 at 24 months (N = 2), due to β-amyloid plaques in cerebellum. PVEC reduced the discrepancy between SUVR-PET and autoradiography from −22% to +2% and increased the differences between young and aged transgenic animals. SUVR and plaque load correlated highly between strains for uncorrected (R = 0.94, p<0.001) and PVE-corrected (R = 0.95, p<0.001) data. We find that APPswe/PS2 mice may be optimal for longitudinal amyloid-PET monitoring in planned interventions studies.