Normal Rodent Chow Research Articles

The Effects of 5-Hydroxytryptophan on Pain Following Formalin Administration

The aim of the study was to investigate the effect of repeated administration of 5-Hydroxytryptophan (5-HTP) on pain sensation. The quantity of 5-HTP reaching the Central Nervous System (CNS) is affected by the extent to which 5-HTP is converted to serotonin in the periphery. The formalin test was used to access pain perception using two groups of mice weighing 22g-32g (n=10 each). The control group received (15g) normal rodent chow, while the test group received (15g w/w) 5-HTP diet. Daily food intake, water intake and body weight change were measured. The results in the formalin test, showed that the frequency and duration of paw attention and paw lick in both phases of the test was significantly lower (P< 0.05) in the 5-Hydroxytryptophan group (5-HTP) compared to the control. Therefore, repeated administration of 5-Hydroxytryptophan diet decreases pain perception in mice.

Activation of the Mineralocorticoid Receptor Stimulates Protein Disulfide Isomerase: Role in Sickle Cell Disease

Activation of the mineralocorticoid receptor (MR), a critical component of the Renin-Angiotensin-Aldosterone (ALDO)-System (RAAS), has been shown to play an important role in inflammatory and vascular endothelial responses in addition to its well-described effects on sodium and water homeostasis. Activationof endothelial cells leads to, among other factors, increased endothelin-1 (ET-1) and protein disulfide isomerase (PDI) release. PDI and ET-1 contribute to vascular inflammation and are increased in patients with SCD and sickle transgenic mouse models. The MR is a member of the steroid family of nuclear receptors and transcription factors that upon activation binds to hormone response elements of edn1, the gene for ET-1, leading to increased ET-1 expression.In vivo, blockade of MR has been shown to reduce circulating ET-1 levels and ET-1 mRNA expression. However, the role of MR in SCD is unclear. We hypothesized that MR blockade in sickle transgenic mice would reduce PDI activity and improve hematological parameters and inflammation. We first studied EA.hy926 (EA) cells, a human endothelial cell line that expresses MR. We incubated EA cells with ALDO (10-8 M), an MR agonist, for 24 hr and observed a rise in PDI mRNA levels by qRT-PCR (P<0.01, n=5), an event that was blocked by pre-incubation of EA cells with 1 μM canrenoic acid (CA), an MR antagonist (P<0.05, n=5). We then measured PDI activity in the supernatant of ALDO-stimulated EA cells using a Di-E-GSSH fluorescent marker and observed a rise in PDI activity following ALDO (10-8 M) when compared to vehicle treatment (P<0.05; n=5). To test the in vivo effects of MR activation, we studied Berkeley Sickle Transgenic (BERK) mice that were randomized to receive either normal rodent chow or chow containing eplerenone (156 mg/kg per day), an MR antagonist (MRA), for 14 days. We observed significantly lower plasma PDI activity in mice treated with MRA than those on regular chow (63.7 ± 8.7 control diet to 47.9 ± 2.4 eplerenone, Relative Fluorescence Units (RFU); P<0.005, n=6 and 9, respectively). Treatment with MRA was associated with reduced plasma ET-1 and myeloperoxidase (MPO) levels in BERK mice. We also studied RBC Gardos channel activity in these mice and observed a significant reduction in clotrimazole-sensitive K+ efflux following MR blockade (2.49±0.5 control and 1.37±0.3 mmol/1013 cells x hr; P<0.04 n= 5 and 7 respectively). Consistent with these results, MR blockade was associated with increases in both erythrocyte MCV (41.3±2.5 vs 47.4±1.1 fL, P<0.03, n=7) and reticulocyte MCV (53.6.3±2.8 vs 60.1±0.6 fL, P<0.02, n=7). We also studied gene expression by qRT-PCR in heart tissue from these mice and observed that MR blockade reduced mRNA expression of: ET-1 (0.654 ± 0.233, P<0.05, n=5 and n=7); PDI (0.546 ± 0.063, P<0.01, n=5 and n=7); and Tumor Necrosis Factor Receptor Superfamily Member 1A mRNA (0.464 ± 0.061, P<0.01, n=5 and n=7). Thus, our results suggest a novel role for RAAS and, in particular, MR activation in SCD. DisclosuresNo relevant conflicts of interest to declare.

EFFECTS OF LONG-TERM ADMINISTRATION OF COOKED BEANS (Vigna unguiculata) DIET ON LEARNING AND MEMORY IN MICE

Purpose: The neurotransmitter, serotonin has neurobehavioural actions which include mood, memory, learning and sleep. Beans, the stable diet of Nigerians contains serotonin and its precursor, 5-Hydroxytryptophan in significant amounts. It was therefore the aim of this study to find out whether long term consumption of cooked beans (Vigna unguiculata) diet has effects on some neurobehavioural parameters notably; learning and memory, using Swiss white mice as experimental animals.Method: Thirty (30) CD1 mice were randomly assigned into three groups, viz; (10, control, 20 Test) weighing 15-30g were fed normal rodent chow, 50% cooked beans diet (w/w) respectively and serotonin precursor (5-HTP) diet (0.2mg/50g w/w) for thirty days. All the mice had access to clean drinking water ad libitum. Before the neurobehavioural parameters were assessed, the phytochemical analysis of the beans, LD50 of the beans (Vigna unguiculata) and that of the serotonin precursor (5-HTP) were determined. Serotonin concentration was measured in the beans using gas chromatography analysis. Learning and memory, was investigated alongside food and water intake and body weight change. Involvement of serotonin pathway was investigated using the set of mice administered serotonin precursor for comparison with the beans diet fed mice. Mice were tested in the Morris water maze for 8 days: 3 days of acquisition training, 3 days of reversal training, 1 day of single probe trial and 1 day for visible platform task.Results: Learning on days 1, 2 &amp; 3 of acquisition training was improved in the cooked beans and serotonin precursor diet-fed mice when compared to control (p&lt;0.001, respectively). The trend was similar during the reversal training, memory was improved in the beans and serotonin precursor diet-fed mice when compared to control (p&lt;0.001). During the probe trial, the swim duration in the South-East quadrant was significantly higher for cooked beans and serotonin precursor group compared to control (p&lt;0.05). However, during the visible platform task, the swim latencies for the cooked beans and serotonin precursor group was significantly lower compared to control. Our finding therefore suggests that long term consumption of beans diet enhances learning and memory. Conclusion and Recommendation: In conclusion, long term consumption of cooked beans diet enhances learning and memory. If this research can be extrapolated to humans, we recommend that the serotonergic potential of beans may be harnessed in the prevention, treatment and management of behaviour/ mental disorders as this could potentially open up a vista for improved mental health status in populations globally.

Effect of Chronic Consumption of Powdered Tobacco (Snuff) on Pain Sensation in Mice.

Pain may be defined as an unpleasant sensory and emotional experience, associated with actual or potential tissue damage. The leaves have social and ethnomedical applications. Tobacco is used traditionally to treat arches, snake bites, bowel complains, convulsions, nervous ailment and to cure/heal the pain of toothache when chewed (Melson, 1944). Therefore this study was designed to investigate the effect of chronic consumption of powdered tobacco on pain sensation using two groups of Swiss mice (control and test) weighing 18g-28g (n=15 each).The control received 100g of normal rodent chow, while the test group received 1g of powdered tobacco in 99g of rodent chow per day. Water was given ad libitum while daily food and water intake, as well as body weight changes, were monitored during the 31-day study. The tail flick and formalin tests were used to assess pain sensation. The results showed that the latency of tail flick did not differ between the test and control group. However, in the formalin test, the frequency of paw attention in both phases of the test was significantly lower (P< 0.05, 0.01) compared to the control group. The duration and frequency of grooming (P<0.05, 0.01) and SAP (P< 0.01) were also significantly lower in the tobacco diet group compared to the control in the early phase, thus, showing decrease pain sensation. Therefore, chronic consumption of powdered tobacco diet may decrease pain sensation.

Connecting Female Infertility to Obesity, Inflammation, and Maternal Gut Dysbiosis.

The prevalence of obesity has reached alarming proportions globally, and continues to rise in both developed and developing countries. At least 2.8 million people each year die as a result of being overweight or obese. According to the estimates by the World Health Organization in 2008 more than 1.4 billion adults were overweight in 2008, and more than half a billion were obese, and the number of overweight and obese adults is projected to increase to 2.2 billion and 1.1 billion, respectively, by 2030. As the numbers of obese women increases the associated comorbidities like type 2 diabetes, coronary heart disease, cancers and reproductive disorders will also increase. Women with obesity have a number of reproductive disorders including problems with ovulation, decreased rates of conception, infertility, early pregnancy loss, birth defects, and reduced assisted reproductive technology success (1, 2). The increased incidence of miscarriages and assisted reproductive technology failure in obese women (3, 4) is attributed in large part to poor oocyte quality manifest as impaired oocyte maturation and disrupted spindle morphology. A number of investigators provide evidence of metabolic perturbations in oocytes from obese mothers including high levels of intracellular lipid (5), elevated endoplasmic reticulum (6), and oxidative stress (7, 8), increased accumulation of oocyte mRNA (9), and mitochondrial dysfunction (1, 7). These obesity-induced alterations in ovarian follicle development and oocyte quality are associated with activation of systemic or local inflammatory pathways (10–12). In this issue of Endocrinology, Xie et al (13) find that obesity-dependent increases in proinflammatory signaling regulates the abundance of oocyte specific mRNAs (Figure 1). This journey began when this group found that a subset of maternal RNAs, including maternal effect genes, are elevated in oocytes from mature obese mice (9) and women with metabolic dysfunction (14). These observations are important because the increased abundance of maternal effect genes is known to interfere with normal embryonic development (15, 16). In the present study, to induce the obese phenotype the investigators fed young adult female mice normal rodent chow or a high-fat diet containing 45% or 60% of its calories from fat. The highfat diet induced increases in adipose tissue and whole body

Altered metabolism and resistance to obesity in long-lived mice producing reduced levels of IGF-I.

The extension of lifespan due to reduced insulin-like growth factor 1 (IGF-I) signaling in mice has been proposed to be mediated through alterations in metabolism. Previously, we showed that mice homozygous for an insertion in the Igf1 allele have reduced levels of IGF-I, are smaller, and have an extension of maximum lifespan. Here, we tested whether this specific reduction of IGF-I alters glucose metabolism both on normal rodent chow and in response to high-fat feeding. We found that female IGF-I-deficient mice were lean on a standard rodent diet but paradoxically displayed an insulin-resistant phenotype. However, these mice gained significantly less weight than normal controls when placed on a high-fat diet. In control animals, insulin response was significantly impaired by high-fat feeding, whereas IGF-I-deficient mice showed a much smaller shift in insulin response after high-fat feeding. Gluconeogenesis was also elevated in the IGF-I-deficient mice relative to controls on both normal and high-fat diet. An analysis of metabolism and respiratory quotient over 24 h indicated that the IGF-I-deficient mice preferentially utilized fatty acids as an energy source when placed on a high-fat diet. These results indicate that reduction in the circulating and tissue IGF-I levels can produce a metabolic phenotype in female mice that increases peripheral insulin resistance but renders animals resistant to the deleterious effects of high-fat feeding.

Influence of Long Term Administration of Nevirapine on Serum Liver Enzymes Profile in Albino Wistar Rats

The effect of long term administration of nevirapine (NVP) on serum levels of some liver enzymes in albino Wistar rat was investigated. A group of rats treated with NVP (0.4 mg/kg body weight) for 2 weeks thereafter, two times daily in addition to normal rodent chow for 12 weeks were examined for serum levels of aspartate transaminase, alanine aminotransferase, alkanine phosphatase, gamma glutamyl transferase. The levels of aspartate transaminase (AST), alanine aminotransferase (ALT), alkanine phosphatase (ALP) and gamma glutamyl transferase (GGT) for the NVPtreated group were significantly (p < 0.001) higher as compared to their control(s), respectively. In conclusion, results of the study indicate that long term administration of NVP may be injurious to liver cell integrity, and this might impair liver function in the rat. If this result is applicable to humans, long term administration of NVP could hamper the integrity and function of the liver in individual users.

In vivo noninvasive measurement of skin autofluorescence biomarkers relate to cardiovascular disease in mice

The formation of reactive oxygen species (ROS) is associated with cardiovascular disease (CVD). High dietary cholesterol can significantly alter the delicate balance between pro-oxidation and antioxidant defences leading to reactive oxygen species formation in the vasculature, without significant structural changes in tissue composition. We aimed to establish a methodology for the noninvasive assessment of skin fluorescent biomarkers in mice. C57/black/6 wild-type (WT; n = 25) male mice were subdivided to receive normal rodent chow (n = 11) or a high cholesterol diet (2% cholesterol; n = 14) for 20 weeks. Skin autofluorescence measurements were made on the backs of anaesthetized (1.5-2% isoflurane in oxygen) mice. A laser probe was used to make simultaneous measurements of: collagen, elastin, nicotinamide pyridoxine, flavins, lipofuscin and β-carotene. Results are expressed as group mean in arbitrary units (AU) ± standard error (SE). Hearts were excised and weighed (mg); cardiac hypertrophy was measured by ratio [heart weight (mg)/bodyweight (g) ± SE]. Student's t-test was used for statistical significance analysis (p ≤ 0.05). There were no significant differences between cholesterol- and chow-fed animals for collagen (34 ± 5AU vs. chow 34 ± 4 AU, p = 0.51) and elastin (66 ± 6 AU vs. chow 82 ± 7 AU, p = 0.11). Significant differences were evident for nicotinamide adenine dinucleotide (92 ± 7 AU vs. chow 118 ± 7 AU, p = 0.01), pyridoxine (56 ± 4 AU vs. chow 73 ± 4 AU, p = 0.01), flavins (44 ± 3 AU vs. chow 57 ± 4 AU, p = 0.01), lipofuscin (35 ± 3 AU vs. chow 46 ± 3 AU, p = 0.01) and β-carotene (19 ± 2 AU vs. chow 25 ± 2 AU, p = 0.01). Cholesterol-fed animals had significantly heavier hearts (7 ± 0.3 ratio vs. chow 5 ± 0.1 ratio, p = 0.001). Cholesterol feeding induced cardiovascular disease as noted by cardiac hypertrophy in wild-type mice. A reduction was observed in pyridoxine, nicotinamide adenine dinucleotide, flavins, lipofuscin and β-carotene, which are established risk factors for cardiovascular disease. We report no significant changes in structural proteins collagen and elastin, suggesting no generalized tissue restructuring, which might otherwise explain the observed pathological differences.

Effect of Nevirapine Administration on Biliary Secretion/its Biochemical Composition in Albino Wistar Rats

Background: Nevirapine is an antiretroviral medication that prevents human immunodeficiency virus cells from multiplying in the blood. This study was undertaken to ascertain whether nevirapine administration affects bile secretion in albino Wistar rats. Methods: Male and female albino Wistar rats (n=20, 50-125 g body weight) at the start of the experiment were used for the study. Rats in the control group (n=10) were administered normal saline (0.4 mg/kg body weight) + normal rodent chow, whereas the nevirapine group (n=10) were fed by gavage nevirapine (0.4 mg/kg body weight) two times daily (07:00 h and 18:00 h) in addition to normal rodent chow for 12 weeks. All animals were allowed free access to clean drinking water. Biliary secretion, cholesterol, bilirubin, conjugated and unconjugated bilirubin levels, and bile electrolytes were measured. Results: Biliary secretion in the nevirapine-treated group was significantly lower (p<0.05) than in the control group. Total cholesterol, total bilirubin and unconjugated bilirubin were significantly higher (p<0.001) in the nevirapinetreated group when compared to control. Conjugated bilirubin was also increased in the nevirapine-treated group though not statistically different from the control. Electrolytes (sodium and chlorine) content of bile were significantly lower (p<0.01 and p<0.001) in the nevirapine-treated group when compared to control. However, (potassium and bicarbonate) content of bile were significantly higher (p<0.05 and p<0.001) in the nevirapine-treated group when compared to control. Conclusion: Long term administration of nevirapine may lead to reduction in biliary secretion, increase bilirubin/ cholesterol levels and alter bile electrolytes’ composition. This implies that NVP may provoke liver damage.

Morphology of the Small Intestine of Albino Wistar Rats Following Long Term Administration of Nevirapine

Background: Nevirapine (NVP) is an antiretroviral medication that prevents human immunodeficiency virus (HIV) cells from multiplying in the blood. This study was undertaken to ascertain whether NVP administration affects intestinal morphology using albino Wistar rats. Materials and methods: Sixty adult albino Wistar rats were used for the study. Rats in the control group (n=30) were fed normal rodent chow, while the NVP group (n=30) were fed by gavage NVP (0.4 mg/kg body weight) twice daily (7:00 am and 6:00 pm) in addition to normal rodent chow for 12 weeks. All animals were allowed free access to clean drinking water. Morphological examination of tissues (duodenum, jejunum and ileum) was done. Results: Gross morphology of the duodenum in the NVP-treated group showed hypertrophy of the Bruner’s glands within the sub-mucosa as compared to control where the tissues appeared intact. Gross morphology of the jejunum in the NVP-treated group showed hyperplasia of mucosal cells and mild desquamation of epithelia, when compared to control the tissues appeared intact. Gross morphology of ileum in the NVP-treated group showed reductions in the density of Payer’s patches and diffused areas of necrosis of mucosal epithelium when compared to control where tissues appeared intact. Conclusion: From the result of the study, long term administration of NVP may cause disorganization of the

Aldosterone Stimulates Neutrophils Leading To Increased β-Glucuronidase, Protein Disulfide Isomerase and Myeloperoxidase Secretion

Aldosterone (ALDO) has been shown to play an important role in inflammatory responses in addition to its well described effects on sodium homeostasis via activation of the mineralocorticoid receptor (MR). However, its effects on polymorphonuclear leukocytes (PMNC) are not well described. We isolated untouched circulating human PMNC by immunomagnetic isolation following density gradient sedimentation with PolymorphPrep from otherwise healthy subjects. Flow cytometric analyses showed greater than 97% of PMNC were positive for the myeloid-neutrophil markers, CD45, CD16 and CD66b. We show that PMNC express MR by western blot and RT-PCR analyses. We incubated PMNC with ALDO (10–9–10–7M) for 30 min and observed a dose-dependent rise in β–glucuronidase release with an EC50 of 6.11 nM (P<0.001, n=3), an event that was blocked by pre-incubation of cells with 1μM canrenoic acid (CA), an MR antagonist (P<0.04, n=3). In addition, our results show that incubation of human PMNC with 10-8M ALDO likewise led to increases in myeloperoxidase ([MPO], P<0.05, n=3) and protein disulfide isomerase ([PDI], P<0.01, n=4), a multifunctional enzyme of the thioredoxin superfamily that mediates redox modifications, regulates KCNN4 channel and erythrocyte volume and is up-regulated under hypoxic conditions (Prado, 2013 FASEB J). We then studied the effects of ALDO on HL-60, a human promyelocytic cell line, induced to differentiate into neutrophil-like cells by incubation for 5 days with 1.3% DMSO. Our results likewise show an increase in MPO responses upon 10–8M ALDO stimulation as compared to vehicle (AUC: 1090±147 to 505±48, P<0.02, n=3). We have recently reported that aldosterone stimulates increases of striatin, a scaffolding protein that interacts with caveolin-1, and co-precipitates with striatin and as such may facilitate cross talk of signaling complexes. As there are no pharmacological inhibitors of striatin we used a molecular approach to reduce striatin levels. In differentiated HL-60 cells, siRNA against striatin led to reduced MPO responses (AUC: 590±14 to 528±13, P<0.05, n=3) that were associated with significantly reduced striatin mRNA levels but not when cells were transfected with scrambled siRNA as determined by quantitative RT-PCR with ABI TaqMan detection probes and β-microglobulin used as an endogenous control (P<0.01, n=3). These results suggest that striatin plays an important role in ALDO-stimulated degranulation responses. Of importance we also observed that incubation with ALDO (10–9–10–7M) in differentiated HL60 cells led to increases in the oxidative-respiratory burst [superoxide production] in a dose- and time-dependent manner (P<0.01, n=4). Consistent with these results, we observed that ALDO likewise led to significant increases in the oxidative-respiratory burst in human PMNC (P<0.01, n=3). As there is evidence that activated neutrophils, MPO and PDI are elevated in Sickle Cell Disease, we studied the in vivo effects of MR blockade in BERK sickle transgenic mice, a model of increased oxidative stress. Sickle mice were randomized to receive either normal rodent chow or chow containing eplerenone (156 mg/kg per day), an MR receptor antagonist, and tap water ad libitum for 14 days at which time the mice were sacrificed and blood collected. We observed that mice on eplerenone had significantly lower plasma PDI activity than mice on regular chow (63.7 ± 8.7 control diet to 47.9 ± 2.4 eplerenone, Relative Fluorescence Units [RFU]; P<0.005, n=6 and 9) and lower MPO levels (AUC: 214±11 to 73±20, P<0.03, n=3); events that were associated with increases in both erythrocyte MCV (41.3±2.5 vs 47.4±1.1 fL, P<0.03, n=7) and reticulocyte MCV (53.6.3±2.8 vs 60.1±0.6 fL, P<0.02, n=7). Thus, our results suggest that MR activation by ALDO is a novel mechanism for neutrophil stimulation and as such represents a novel therapeutic target aimed at ameliorating the vascular complications of Sickle Cell Disease. Supported by NIH R01HL090632 (AR) and R01HL096518 (JRR). Disclosures:No relevant conflicts of interest to declare.

Maternal choline supplementation improves spatial learning and adult hippocampal neurogenesis in the Ts65Dn mouse model of Down syndrome

In addition to intellectual disability, individuals with Down syndrome (DS) exhibit dementia by the third or fourth decade of life, due to the early onset of neuropathological changes typical of Alzheimer's disease (AD). Deficient ontogenetic neurogenesis contributes to the brain hypoplasia and hypocellularity evident in fetuses and children with DS. A murine model of DS and AD (the Ts65Dn mouse) exhibits key features of these disorders, notably deficient ontogenetic neurogenesis, degeneration of basal forebrain cholinergic neurons (BFCNs), and cognitive deficits. Adult hippocampal (HP) neurogenesis is also deficient in Ts65Dn mice and may contribute to the observed cognitive dysfunction. Herein, we demonstrate that supplementing the maternal diet with additional choline (approximately 4.5 times the amount in normal rodent chow) dramatically improved the performance of the adult trisomic offspring in a radial arm water maze task. Ts65Dn offspring of choline-supplemented dams performed significantly better than unsupplemented Ts65Dn mice. Furthermore, adult hippocampal neurogenesis was partially normalized in the maternal choline supplemented (MCS) trisomic offspring relative to their unsupplemented counterparts. A significant correlation was observed between adult hippocampal neurogenesis and performance in the water maze, suggesting that the increased neurogenesis seen in the supplemented trisomic mice contributed functionally to their improved spatial cognition. These findings suggest that supplementing the maternal diet with additional choline has significant translational potential for DS.

805 Knockout of SULF2 Significantly Ameliorates Hypercholesterolemia, Steatohepatitis and Liver Fibrosis in Obese Mice Fed a Fast Food Diet

Background: Nonalcoholic fatty liver disease (NAFLD), an important consequence of metabolic disorders such as obesity and type 2 diabetes (T2DM), has become highly prevalent in the US, and is now an important public health concern. Recent reports have identified the heparan sulfate degrading endosulfatase sulfatase 2 (Sulf2) as a suppressor of triglyceriderich lipoprotein (TRL) remnant clearance by hepatocytes in T2DM mice, thus enhancing postprandial hypertriglyceridemia. Further, inhibition of Sulf2 normalized the VLDL-binding capacity of hepatocytes from T2DM mice and abolished postprandial hypertriglyceridemia. We hypothesized that an increase in hepatic Sulf2 occurring during insulin resistance states such as obesity and T2DM decreases hepatic clearance of TRL remnants and contributes to the development of NAFLD, which can be characterized by non-alcoholic steatohepatitis (NASH) and liver fibrosis. Aims: To investigate (1) whether a fast food diet high in saturated fats, cholesterol, and fructose can produce insulin resistance and overexpression of Sulf2; and (2) whether depletion of Sulf2 can protect against NASH and liver fibrosis in obese mice on a fast food diet. Methods: Sulf2 knockout and wild-type mice (n=12 each) were fed a fast food diet rich in saturated fats and cholesterol along with fructose in their drinking water or normal rodent chow with tap water, beginning at 6-8 weeks of age. At 9 months, mice were sacrificed, blood was obtained for assessment of insulin resistance, alanine transaminase (ALT) and serum lipids, and the livers were harvested and examined for steatosis, steatohepatitis, and fibrosis. Results: The fast food diet produced obesity and insulin resistance. Sulf2 knockout mice fed the fast food diet had significantly less weight gain and lower serum ALT, total cholesterol and LDL-cholesterol than wild-type mice (p,0.05). The fast food diet also induced histopathological features of NASH, including hepatic microvesicular steatosis, inflammation and fibrosis. Hepatic fibrosis was significantly decreased in Sulf2 knockout mice compared to controls (p,0.05). Conclusions: Depletion of Sulf2 improves hypercholesterolemia, steatohepatitis and hepatic fibrosis in a mouse model of NASH induced by a fast food diet. Inhibition of SULF2 may be a potential preventive and therapeutic strategy for NASH with progressive fibrosis.

Ulcerogenic and intestinal motility/transit stimulating actions of nevirapine in albino Wistar rats

The antiretroviral is a non-nucleoside reverse transcriptase inhibitor of human immunodeficiency virus type 1. This study was undertaken to investigate the effect of nevirapine (NVP) administration on gastric acid secretion, pepsin secretion, mucosal secretion, intestinal motility, and transit using apparently healthy albino Wistar rats. Eighty albino Wistar rats (50-125 g body weight) from the start of the experiment were used for the study. Rats in the control group were fed normal rodent chow, while the NVP group was fed by gavage NVP (0.4 mg/kg body weight) two times daily (07:00 and 18:00 hours) in addition to normal rodent chow for 12 weeks. All animals were allowed free access to clean drinking water. Mean basal gastric output and peak acid output following histamine administration in the NVP-treated group were significantly higher (p < 0.001, respectively) compared to the control. Following cimetidine administration, there was significant decrease (p < 0.001) in peak acid output in the NVP-treated group compared to the control. The concentration of gastric pepsin, adherent mucus secretion, and mean value for ulcer score were significantly higher (p < 0.001) compared to their control group, respectively. There were significant increases (p < 0.05, respectively) in intestinal motility and basal contraction (p < 0.05) and increase in intestinal transit of the ileum of NVP-treated rats compared to their control, respectively. Results of the study suggest that NVP administration might provoke gastric ulceration in rats which may be caused by high pepsin, high basal acid output, and increased intestinal motility and transit.

Dietary xenosterols lead to infertility and loss of abdominal adipose tissue in sterolin-deficient mice

The investigation of the human disease sitosterolemia (MIM 210250) has shed light not only on the pathways by which dietary sterols may traffic but also on how the mammalian body rids itself of cholesterol and defends against xenosterols. Two genes, ABCG5 and ABCG8, located at the sitosterolemia locus, each encodes a membrane-bound ABC half-transporter and constitutes a functional unit whose activity has now been shown to account for biliary and intestinal sterol excretion. Knockout mice deficient in Abcg5 or Abcg8 recapitulate many of the phenotypic features of sitosterolemia. During the course of our studies to characterize these knockout mice, we noted that these mice, raised on normal rodent chow, exhibited infertility as well as loss of abdominal fat. We show that, although sitosterolemia does not lead to any structural defects or to any overt endocrine defects, fertility could be restored if xenosterols are specifically blocked from entry and that the loss of fat is also reversed by a variety of maneuvers that limit xenosterol accumulation. These studies show that xenosterols may have a significant biological impact on normal mammalian physiology and that the Abcg5 or Abcg8 knockout mouse model may prove useful in investigating the role of xenosterols on mammalian physiology.

Blockade of the Mineralocorticoid Receptor in Sickle Transgenic Mice Reduces Circulating Protein Disulfide Isomerase and Gardos Channel Activity

AbstractAbstract 999Endothelin-1 (ET-1), erythrocyte sickling and endothelial cell activation have been proposed as important contributors to the pathophysiology of sickle cell disease (SCD). We have provided evidence for the use of ET-1 receptor antagonists in improving hematological parameters in two transgenic mouse models of SCD (Rivera A., 2008, Amer J Physiol). However, the mechanisms that mediate the interplay between red blood cells (RBC) and the endothelium in SCD remain unresolved. Activation of endothelial cells leads to, among other factors, increased levels of protein disulfide isomerase (PDI). PDI catalyzes disulfide interchange reactions, mediates redox modifications and has been observed to be up-regulated under hypoxic conditions. We now report that circulating PDI activity is increased in BERK sickle transgenic mice when compared to wild-type controls. The mineralocorticoid receptor (MR) is a member of the steroid family of nuclear receptors that function as a transcription factor that upon binding to the hormone responsive element of genes such as edn1, the gene for ET-1, leads to increased ET-1 expression. In vivo, blockade of MR has been shown to reduce circulating ET-1 levels and kidney ET-1 mRNA expression. We hypothesized that MR blockade of BERK sickle transgenic mice would lead to reduced PDI activity and improved hematological parameters. Sickle mice were randomized to receive either normal rodent chow or chow containing eplerenone (156 mg/kg per day), an MR receptor antagonist, and tap water ad libitum for 14 days at which time the mice were sacrificed and tissues and blood collected. Plasma PDI activity was calculated by optimization of fluorescently labeled GSSG conversion to GSH. We observed that mice on eplerenone had significantly lower plasma PDI activity than mice on regular chow (63.7 ± 8.7 control diet to 47.9 ± 2.4 eplerenone, Relative Fluorescence Units; P<0.005, n=6 and 9, respectively). We also studied RBC Gardos channel activity in these mice and observed a significant reduction in clotrimazole-sensitive K+ efflux following MR blockade (2.49±0.5 control and 1.37±0.3 mmol/1013 cells × hr; P<0.04 n= 5 and 7 respectively). MR blockade was associated with increases in both erythrocyte MCV (41.3±2.5 vs 47.4±1.1 fL, P<0.03, n=7) and reticulocyte MCV (53.6.3±2.8 vs 60.1±0.6 fL, P<0.02, n=7) as determined by an ADVIA 120 hematology analyzer. In contrast no significant effects on MCHC levels were observed under these conditions. We then studied ET-1 gene expression using quantitative RT-PCR with ABI Taqman chemistries and GAPDH and β-actin as endogenous controls. We observed that MR blockade was associated with reduced expression of ET-1 mRNA in heart tissue (0.654 ± 0.233, ΔΔCT, relative to mice on regular chow, P<0.04, n=5 and n=7) but not lung tissue. Western blot analyses in membranes from human and mouse sickle erythrocytes and endothelial cells revealed the presence of both MR and PDI proteins. We then studied the effects of ET-1 in early cultures of BERK mouse aortic endothelial cells (MAEC). We observed that stimulation of MAEC cells with 100nM ET-1 for 4 hr was associated with increased mRNA expression of PDI levels that was 1.71 fold greater than vehicle treated cells (n=4, P<0.05). Thus, our results suggest that MR blockade reduces ET-1 levels leading to reduced Gardos and PDI activity in Sickle mice. These effects on PDI activity and Gardos channel regulation may represent a novel mechanism for protective effects of MR blockade aimed at ameliorating vascular complications of Sickle Cell Disease. Supported by NIH R01HL090632 to AR. Disclosures:No relevant conflicts of interest to declare.

Effect of sinomenine on vascular smooth muscle cell dedifferentiation and neointima formation after vascular injury in mice

Sinomenine, a pure alkaloid extract from Sinomenium acutum, has anti-inflammatory and immunoregulatory functions. This study investigated the efficiency and the signalling pathways involved in the effect of sinomenine on vascular smooth muscle cell (VSMC) dedifferentiation in response to platelet-derived growth factor (PDGF)-BB stimulation and vascular injury. VSMCs were isolated from rat aorta and preincubated with sinomenine before being stimulated with PDGF-BB. WST and BrdU incorporation assays were used to evaluate VSMC proliferation. Flow cytometric analysis was performed for testing the cell cycle progression. The cell migration of VSMCs were analysed using a Transwell system. The expression of VSMC specific genes and signalling proteins were tested by Western blot. For the animal study, C57/BL6 mice were fed either normal rodent chow diets or sinomenine chow diets that supplemented with 0.09 % sinomenine (w/w) in the normal chows for 14 days before carotid artery wire injury. PDGF-BB activated the dedifferentiation of VSMCs characterised by decreased expression of SMA, Smoothelin and SM22α. However, sinomenine treatment preserved the dedifferentiation in response to PDGF-BB. The activations of mitogen-activated protein kinase extracellular signal-regulated kinases, Akt, GSK3β and STAT3 induced by PDGF-BB were also inhibited in sinomenine-treated VSMCs. In vivo evidence with wire-injured mice exhibited a reduction in neointimal area and an increase in smooth muscle-specific gene expression in the sinomenine-treated group. In this study, we found that sinomenine-suppressed VSMC phenotype switching induced by PDGF-BB in vitro and neointimal formation in vivo. Therefore, sinomenine is a potential candidate to be used in the treatment of vascular proliferative disease.

Decreased immunoreactivity of the polypeptide precursor pro-opiomelanocortin (POMC) and the prohormone convertase pc1/3 after chronic ethanol exposure in Sprague-Dawley rats.

The melanocortin (MC) peptides and opioid peptide β-endorphin are cleaved from the polypeptide precursor pro-opiomelanocortin (POMC). POMC-derived peptides are generated by extensive posttranslational processing that involves several enzymes including prohormone convertase 1/3 and 2 (PC1/3 and PC2). Because ethanol (EtOH) decreases POMC mRNA levels, we determined whether the exposure to an EtOH-containing diet (ED) would significantly reduce central immunoreactivity (IR) of POMC, PC1/3, PC2, and β-endorphin. Male Sprague-Dawley rats were given 18 days of access to a normal rodent chow or a control diet (CD), or short-term (4 days) or long-term (18 days) access to an ED. At the end of the study, rats were perfused with 4% paraformaldehyde, and their brains were sectioned into sets for processing with POMC, PC1/3, PC2, and β-endorphin IR. Rats exposed to an ED for 18 days (ED18) exhibited significant reductions of POMC and PC1/3 IR in the arcuate nucleus of the hypothalamus (Arc) relative to rats pair-fed a CD. On the other hand, rats exposed to an ED did not show any changes of central β-endorphin or PC2 IR relative to rats pair-fed a CD, regardless of length of exposure. Because there were no differences in body weights or caloric intake between the CD and ED groups, reductions of POMC and PC1/3 IR in ED-treated rats are best explained by EtOH exposure rather than altered energy balance. This study shows that EtOH site-specifically reduces POMC and PC1/3 IR in rat brain. These observations are consistent with EtOH-induced reductions of α-melanocyte-stimulating hormone (α-MSH) and POMC IR that were previously reported. As MC agonists have been shown to blunt EtOH intake in rodents, exogenous MC receptor agonists, as well as targets that may increase the synthesis of endogenous α-MSH (e.g., PC1/3), may have therapeutic value for treating alcohol abuse disorders and alcoholism.

Chronic administration of the antiretroviral nevirapine increases body weight, food, and water intake in albino Wistar rats

Nevirapine (NVP) is an antiretroviral medication that prevents human immunodeficiency virus (HIV) cells from multiplying in the blood. This study was undertaken to investigate the effect of chronic administration of NVP on body weight, food, and water intake using apparently healthy albino Wistar rats. Twenty adult albino Wistar rats (50-125 g body weight) were used for the study. Rats in the control group (n=10) were fed normal rodent chow, whereas the NVP group (n=10) were fed by gavage NVP (0.4 mg/kg body weight) two times daily (07.00 h and 18.00 h) in addition to normal rodent chow for 12 weeks. All animals were allowed free access to clean drinking water. Results showed that the mean daily food and water intake in the NVP group were significantly higher (p<0.001) when compared with the control group, respectively. The mean change in body weight in the NVP group was significantly higher (p<0.001) than the control group. These results suggest that chronic administration of NVP may increase body weight in rats, probably due to its stimulatory effects on food and water intake.

Dietary &amp;beta;-glucan Leads to Increased TNF-&amp;alpha; Production in the Lung

Problem statement: Beta (β)-glucan is notable for its ability to stimulate the immune system and as such β-glucan and products containing β-glucan are used as dietary supplements for livestock and companion animals. β-glucan has been shown to activate macrophages and neutrophils and modulate the production of certain cytokines, including the pro-inflammatory cytokine, Tumor Necrosis Factor Alpha (TNF-α). Because TNF-α is a contributing factor in a number of chronic inflammatory diseases and is present at higher concentrations in bronchoalveolar fluid from patients with asthma a preliminary experiment was designed to determine if a diet supplemented with β-glucan leads to increased TNF-α production in response to chitin, an ubiquitous environmental antigen that is associated with airway inflammation. Approach: Mice were divided into two groups. One group was given normal rodent chow and water while the other group was given normal rodent chow and water containing β-glucan (1 mg mL-1) for 14 days. After 14 days, two experimental protocols were conducted to evaluate TNF-α production. In experimental protocol 1, mice were injected intraperitoneally with 4% thioglycollate broth and TNF-α production from the immune cells elicited into the peritoneal cavity was evaluated. In experimental protocol 2, mice were exposed to either chitin or PBS (as a control) via intranasal administration for two consecutive days. Six hours post secondary exposure, Bronchoalveolar Lavage Fluid (BALF) was collected and ELISA for TNF-α performed. Results: TNF-α expression by thioglycollate-elicited cells isolated from animals that consumed β-glucan was greater (27 fold) than controls. Similarly, dietary β-glucan was also associated with increased TNF-α expression (four fold) in the lung, after chitin exposure in vivo. Conclusion: These preliminary results suggest that dietary β-glucan may promote inflammatory responses after exposure to chitin and therefore could be contributing factor to lung inflammation particularly in animals prone to airway inflammatory diseases.