Retro-orbital Blood Samples Research Articles

Effectiveness of Cages Inducing Nasopharyngeal Dysplasia in Wistar Rats (Rattus norvegicus)

The cage is a place used to put experimental animals in experimental studies to study the mechanism of the disease and its response to therapy. Formaldehyde as a carcinogenic compound can increase the occurrence of chronic inflammation which has the potential to increase the growth of cancer cells in experimental animals due to the stress experienced while in the cage. Therefore, the purpose of this study was to compare the effectiveness of large and small cages induced by formaldehyde 30 ppm against nasopharyngeal dysplasia in Wistar rats. This research is an experimental study with a true experimental design with the type of post-test only control group design. This study used 12 male Wistar rats (Rattus norvegicus) which were divided into two groups, namely the group of Wistar rats which were placed in large cages measuring (30 x 24 x 11.5) cm3 of 6 individuals, and in small cages (27 .5 x 21.5 x 9) cm3 of 6 individuals induced by 30 ppm formaldehyde with a span of 6 hours per day for 16 weeks. The rats were terminated and retro-orbital blood samples were taken to measure MDA levels and nasopharyngeal tissue to assess the degree of dysplasia. The results of this study showed that there was a statistically significant difference (p <0.05) between the degrees of nasopharyngeal dysplasia (p=0.003) and MDA levels (p=0.003) in Wistar rats in small cages and large cages. In this study the effectiveness of small cages was greater than large cages in causing dysplasia in the nasopharyngeal tissue of Wistar rats.

Hydro-Ethanol Extract of <i>Persea americana</i> Fruit Pulp Ameliorates Dyslipidaemia and Cardiotoxicity Exerted by Alloxan-Induced Diabetes Mellitus

Diabetes-induced dyslipidaemia has been associated with an increased risk of atherosclerosis and coronary heart diseases. Persea americana fruit has been reported to possess anti-diabetic properties. Therefore, this study assessed the lipid profile and likely cardio-protective effects of hydroethanolic extracts of P. americana fruits in alloxan-induced diabetic Wistar rats. Thirty-five male rats (150 ± 30 g) were divided into 5 groups (n = 7) and treated orally as follows; groups I-II were normal animals treated with distilled water (0.3 ml/day) and P. americana (300 mg/kg) only respectively. Animals in groups III-V were made diabetic using alloxan monohydrate (100 mg/kg i.p.) and treated orally with distilled water (0.3 ml/day), P. americana (300 mg/kg) and glibenclamide (5 mg/kg) respectively for 21 days. Fasting blood glucose level was monitored prior to, after induction of diabetes mellitus, and on day 21 post-treatment, respectively. Thereafter, retro-orbital blood samples were collected after anaesthesia and analysed for insulin, total cholesterol (TC), triglyceride (TG), low density lipoprotein (LDL), high density lipoprotein (HDL) levels, apolipoproteins A1 and B, superoxide dismutase (SOD) and catalase activities, reduced glutathione (GSH), Vitamin C and malondialdehyde levels, respectively. VLDL, atherogenic index (AI) and ApoB/A1 ratio were estimated mathematically. Pancreatic and cardiac structures were also investigated using Haematoxylin and Eosin stains. Treatment with P. ameriacana extracts reduced (p P. americana treated diabetic group. The hydro-ethanol fruit extract of Persea americana attenuates diabetes induced dyslipidaemia and reduces the susceptibility to cardiac impairment in experimental diabetes mellitus.

Possible Cardio-protective Effect of Olive Oil in Experimentally Induced Hyperthyroidism in Rats

Abstract Background Hyperthyroidism is an endocrine disorder that causes both functional and structural changes in the cardiovascular system. Untreated hyperthyroidism and even persistent subclinical thyroid hyperactivity has been associated with the development of Heart failure by inducing atrial fibrillation, chronic hemodynamic overload, oxidative stress and activation of renin-angiotensin pathway. HF secondary to hyperthyroidism is a reversible cause of cardiomyopathy, which highlights the importance of its prompt diagnosis and treatment. Olive oil with its phenolic compounds are powerful antioxidant that may serve as potential therapeutic agents to reduce cardiovascular hazardous effect of hyperthyroidism and intake of virgin olive oil provides benefits on cardiovascular disease. Aim of the work The aim of the present study is to evaluate the possible cardio-protective effects of olive oil, as a natural antioxidant, in experimentally induced hyperthyroidism in rats. Also, to elucidate the underlying mechanism(s), if any, of its effects. Materials and Methods Animals used were 30 adult male albino rats, which were randomly allocated into three groups. Control group(n = 10): received intraperitoneal (i.p.) injection of the solvent of L-thyroxine daily for 4 consecutive weeks. Hyperthyroid group (n = 10): received i.p. injection of L-thyroxine 100μg/kg daily for 4 consecutive weeks. Olive oil-treated hyperthyroid group (n = 10): received i.p. L-Thyroxine as in group II. By the beginning of the third week, 1 ml/100 g B.W extra virgin olive oil was given daily by gavage for 2 weeks. At the end of the study, the overnight fasted rats were subjected to collection of retroorbital blood samples for determination of T3, T4, TSH serum levels. Rats were then weighed and anaesthetized with thiopental sodium (EIPICO, Egypt), IP (40 mg/kg B.W). Then, ECG was recorded. The heart was then subjected to In vitro study of isolated hearts perfused in langendorff preparation. Hearts chambers were then weighed. Apex of left ventricle was used in histopathological examination. Results The induction of hyperthyroidism was confirmed by T3 and T4 levels that were significantly increased, while TSH was significantly decreased in hyperthyroid group as well as olive oil-treated hyperthyroid group compared to control group. Compared to control group, both hyperthyroid and olive oil-treated hyperthyroid groups showed significant decrease in FBW, FBMI, BW% and BMI%. The absolute weights of RV, LV, WH, as well as, their relative weights RV/BW, LV/BW and WH/BW all were significantly increased. Compared to hyperthyroid group, olive oil-treated hyperthyroid group showed significant decrease in FBW as well as FBMI. The LV/BW and WH/BW were significantly increased. As regard ECG findings, HR, QRS duration and amplitude were significantly increased in both hyperthyroid and olive oil-treated hyperthyroid groups as compared to control rats. In the hyperthyroid group the PR interval was significantly shortened compared to control group, after olive oil-treatment PR interval increased significantly compared to hyperthyroid group, and became insignificant from control group. The baseline values in isolated perfused hearts study showed, significant increase in spontaneous beating rate in both hyperthyroid and olive oil-treated hyperthyroid groups as compared to control group. Hyperthyroid group showed non-significant decrease in TG/unit time and significant decrease in HRT compared to control group Olive oil-treated hyperthyroid group TG/unit time was non-significantly increased compared to control group and significantly increased compared to hyperthyroid group. HRT was the same as hyperthyroid group while. Isoprotrenol infusion showed early toxicity in hyperthyroid group indicated by reduced TG/unit time and HRT compared to control group. Olive oil-treated hyperthyroid group TG/unit time maximal response was significantly increased compared to hyperthyroid group, being non-significant from control group. The hematoxylin and eosin study of the left ventricular cardiac muscle, the hyperthyroid group showed apparent structural changes that were partially reversed by olive oil treatment. Conclusion Olive oil was able to impose partial improvement on cardiac systolic function at baseline condition, also, in response to the beta adrenergic agonist, isoproterenol, even though this improvement did not reach control levels.

Atypical Antipsychotic Lumateperone Effects on the Adrenal Gland With Possible Beneficial Effect of Quercetin Co-administration.

Schizophrenia remains one of the most chronic and highly disabling mental disorders. Lumateperone is a recent FDA-approved atypical antipsychotic drug for the treatment of schizophrenia. However, the internal FDA pathologist raised concerns regarding pigment deposition associated with degeneration in different tissue in animal studies with lumateperone treatment. The adrenal gland may be implicated in lumateperone side effects, and quercetin may have the ability to fulfill this treatment gap. To prove this hypothesis, 40 male guinea pigs were used and divided into four groups; control, quercetin-treated, lumateperone-treated, and quercetin/lumateperone cotreated orally for 28 consecutive days. Behavioral forced swim (FST) and open field (OF) tests were done at the end of treatment. Retro-orbital blood samples were taken to assess hormones: adrenocorticotropic hormone (ACTH), cortisol, dehydroepiandrosterone acetate (DHEA), and aldosterone, along with an assessment of oxidative stress parameters: malondialdehyde (MDA), glutathione (GSH), and superoxide dismutase (SOD). Adrenal glands were extracted for histopathological assessment with H&E, Mallory trichome staining, immunostaining, and electron microscopy studies. Lumateperone-treated group showed a significant reduction in the activity in FST and OF with histopathological deterioration in adrenal secretory function and structure and increased expression of interleukin-6 (IL-6), CASPASE-3, collagen deposition, and decreased proliferating cell nuclear antigen (PCNA). Cytoplasmic vacuolation, pyknosis of the nuclei, increase in the lysosome, lipofuscin pigment, and cellular infiltration with diminishing in the number of secretory granules could all be observed in lumateperone-treated group. Coadministration of quercetin and lumateperone showed improvement of the previously deteriorated parameters. Quercetin had a prophylactic effect against lumateperone depressive-like effect on animal behavior and its possible adrenal damage.Graphical Conceptual framework for the proposed mechanism of action of coadministration of quercetin and lumateperone.

Probable Protective Effect of Exogenous Administration of Growth Hormone on Age –induced Hypercholesterolemia in Rats.

Background: Aging is an inevitable biological process accompanied by many metabolic changes such as hypercholesterolemia that increased risk of cardiovascular disease in elderly. It was reported that growth hormone GH substitution contributes to dyslipidemia improvement by modulating expression of LDL R. To our knowledge, no clarified distinguished mechanisms have been puplished yet . Objectives: To test the hypothesis that GH would improve age induced hypercholesterolemia by over expression of LDL-R m RNA . Also, to distinguish its effects on the inflammatory response and the oxidative state that may clarify the mediating mechanisms. Material& method: thirty male albino rats were divided into three groups (10 rats each): adult control, aging group, GH aged treated groups. At the end of experiment, retro-orbital blood samples were taken for estimation, lipid parameters and serum amyloid a then liver were removed for measurement of tissue catalase and LDL-R mRNA real time. Results: The present study revealed that aging worsened lipid profile. Fortunately GH substitution leads to lipid profile improvement. These metabolic changes might assert to oxidative state and inflammatory response improvement accompanying apparent LDL-R mRNA real time. Conclusion: GH supplementation can positively modulate the lipid profile in aging through modifying effect on the inflammatory response , anti-oxidant state and over expression of hepatic LDL-R . The clinical inferences of these results require further investigations.

The effect of Escherichia Coli induction on superoxide dismutase (SOD) and Malondialdehyde (MDA) levels in acute rhinosinusitis white rats models

Background: Acute Rhinosinusitis is an inflammation of the paranasal sinuses and the nasal cavity lasting no longer than 6 weeks. One of the etiologies is the gram-negative bacteria, the lipopolysaccharide layer containing bacteria Escherichia coli. Lipopolysaccharide was the leading cause of inflammatory mechanisms, and subsequent Reactive Oxygen Species production was thought of as the agent that injure the cells thorough the oxidative stress. This study aimed to determine the effect of E-coli induction on the ratio of SOD and MDA levels in acute rhinosinusitis white rats model.Methods: This study used an experimental post-test only control group design with white rats strain that met the inclusion and exclusion criteria divided into two groups. The study includes 8 rats as control groups and 32 rats in the treatment group. Treatment includes induction of rhinosinusitis by E-coli. SOD and MDA levels measured from retro-orbital venous blood samples were measured as marker oxidative stress on 7th, 14th,21st, and 28th days. Data were analyzed using SPSS version 22 for Windows.Results: There were significant differences in SOD and MDA levels from day 7 to day 28 between the control and the intervention groups (p<0.001). The mean SOD levels on days 7, 14, 21, and 28, were lower than those of the control group. The MDA levels from day 7 to day 28 of the intervention group was significantly higher than that of the control group (p<0.001). Spearman Correlation test obtained a significant correlation between SOD and MDA levels (r = -0.604; p<0.001 on day 7 and r = -0.453; p< 0.003)Conclusion: There is a significant correlation between the effect of induced E coli on SOD and MDA levels in acute rhinosinusitis white rats models.

PSXIII-3 Maternal eicosapentaenoic acid feeding decreases placental and fetal lipid deposition and improves antioxidant capacity through a Sirtuin-1 independent

Abstract This research aimed to evaluate whether maternal dietary n-3 polyunsaturated fatty acids (PUFA) supplementation could decrease placental lipid deposition and improve placental antioxidant capacity through a SIRT1-dependent way. In the present study, male and female SIRT1+/- mice were mated overnight, then primiparous SIRT1+/- mice were fed a 60% kcal high-fat-diet (HFD) or equienergy EPA diet (4.4% EPA-ethyl ester) (n = 22/each group). At pregnant 18.5 d, retro-orbital blood samples were obtained then sacrificed by cervical dislocation. Placentas and its corresponding fetuses were collected by caesarean section. We found that the EPA diet significantly changed maternal plasma, placenta and fetus fatty acid composition, and decreased placental and fetal lipid content. Meanwhile, EPA treatment significantly increased placental and fetal antioxidant capacity, however, lipid peroxidation products malondialdehyde was also increased. Moreover, EPA treatment significantly activated placental UCP1 and PGC1α expression and alleviated mitochondrial swelling induced by HFD. SIRT1 knockout had no significant effect on placental lipid content, but notably decreased fetal triglyceride (TG) and non-esterified fatty acid (NEFA) content. Meanwhile, SIRT1 knockout significantly decreased the ratio of n-3 PUFA in placenta. SIRT1 knockout also significantly decreased PGC1α mRNA and protein level and increased mitochondrial swelling. Both EPA treatment and SIRT1 deficiency had no effect on BODIPY-FL-C16 (a fluorescent labeled palmitic acid) uptake in placenta and fetus. No significant effect was observed between diet and genotype. In conclusion, our study demonstrated that maternal EPA feeding decreased placental and fetal lipid deposition and improved antioxidant capacity through a Sirtuin-1 independent mechanism.

Antithrombin- and Heparin-Binding Exosites Regulate Extravascular Binding and Clearance of Factor IXa

Introduction: Administration of increasing doses of FIX to hemophilia B (HB) mice reveals that the extravascular compartment is capable of binding several-fold more FIX than circulates in the plasma (Cooley et al., Blood 2019). FIX binds rapidly and reversibly to vascular endothelium and the extravascular matrix, in part mediated by interaction of the Gla domain with collagen IV (Col IV). Anticoagulant heparan sulphate and Col IV localize predominantly to the basement membrane and supramolecular structure analysis suggests that heparan sulphate chains are integrated into Col IV-containing networks. Previous work has demonstrated the contribution of the heparin and antithrombin (AT) binding exosites on the protease domain to the regulation of FIXa activity. We now compare the contribution of these protease exosites to in vivo recovery and clearance of the FIX(a) protease and zymogen in hemophilia A (HA) or HB mice. Methods: Recombinant human FIX variants expressed in HEK293 cells with substitutions in the AT- (R150A) and heparin-binding (K126A/K132A) exosites (chymotrypsinogen #) were purified to homogeneity and activated to FIXa with FXIa. FIX(a) pharmacokinetics (PK) in HA or HB mice were determined with injection of a weight-based dose into the tail vein. Serial retro-orbital blood samples were collected, plasma isolated and FIX(a) content determined with a human FIX specific ELISA. FIX(a) concentrations were plotted versus time and fit to the equation: [IX]t = [IX]max*(C1*e(-k1*t)+ C2*e(-k2*t)), where k1and k2are rate constants for the initial and terminal elimination phases, respectively. As the 4-parameter fit resulted in some large error estimates, the two phases were fit separately to estimate the respective parameters. Results: PK for clearance of predicted 30% plasma levels of human FIXa (5 min to 3 hr) or FIX (5 min to 25 hr) were examined in HA (Fig 1) or HB mice (Fig 2), respectively. HA mice were employed for FIXa to avoid in vivoclot formation triggered by active protease. Notably, FIXa plasma clearance was relatively prolonged with ~50% of recovered antigen levels present at 25-30 min post-injection. Similar to FIX, the pattern of FIXa plasma clearance suggested a 2-compartment model with initial and terminal phases of elimination. FIXa WT demonstrated approximately ~23% recovery of predicted levels at 5 min in HA mice, similar to the zymogen (21%) in HB mice (Table I). FIXa R150A (reduced AT affinity) demonstrated markedly enhance recovery (43%) in HA mice relative to WT, while FIX R150A (24%) in HB mice which was similar to WT. FIXa K126A/132A (reduced heparin affinity) demonstrated markedly enhanced recovery (55%) in HA mice, slightly enhanced relative to the similarly increased zymogen recovery (48%) in HB mice. FIXa WT-GGACK (active site inhibited) demonstrated similar recovery to the unmodified FIXa WT with a significantly faster rate of clearance (2.2 fold) in the initial phase (k1) and a significantly slower rate of clearance (3.6-fold) in the terminal phase (k2). Overall, the initial rate (k1) was not significantly different among the active FIXa variants in HA mice or among zymogen variants in HB mice. However, comparison of individual proteases variants to their respective zymogens demonstrated that the initial rate (k1) of plasma clearance was 2-3 fold faster for the proteases, while the terminal rate (k2) was 5-9 fold faster (FIXa R150 had the smallest relative increase). Conclusions: The 2-phase elimination pattern for FIXa suggests that significant amounts of protease may exist in a non-circulating extravascular pool, similar to the zymogen. Further, clearance of FIXa antigen from mouse plasma is prolonged, consistent with the half-life of FIXa activity in human plasma (~41 min). FIXa variants with reduced affinity for AT (R150A) or heparin (K126A/K132A) showed enhanced recovery compared to FIXa WT, suggesting that these protease exosites make independent contributions to the extravascular binding of FIXa. The heparin exosite makes a similar contribution to zymogen binding to extravascular sites, but the AT exosite does not, likely due to being unavailable in the zymogen. Active site inhibited FIXa (GGACK) demonstrates an accelerated initial elimination phase relative to FIXa WT, suggesting re-distribution of the protein is conformation-dependent. The subsequent terminal elimination phase was slowed, consistent with an inability to be inhibited and cleared by AT Disclosures Sheehan: Pfizer: Research Funding; Bioverativ: Consultancy; Genetech: Consultancy.

Ameliorative Effect of Exercise Training on Age-Related Vascular and Biochemical Changes in Rats

AbstractBackground: Physical exercise is suggested as a life style modification to improve the age related patho-physiologic heamodynamic alterations.Aim of Study: The present study was designed to evaluate the possible ameliorating role of aerobic exercise training in improving age associated vascular dysfunction and hemody-namic disturbance.Material and Methods: Thirty male albino rats of local strain were used for this study. Rats were divided into three main groups: The young control (aged 3-4 months, weighing 150-200gm) (n=10), aged sedentary (aged 22-24 months, weighing 300-400gm) (n=10) and aged exercised group (aged 22-24 months, weighing 300-400gm) (n=10). Rats in the aged exercised group practiced moderate intensity treadmill exercise for 5 days/week for 6 weeks. After 6 weeks rats were subjected for rat tail-measurement of SBP. Then, fasting retro-orbital blood samples were collected for measuring serum lipid profile (TC, HDL-C, LDL-C, VLDL-C and TGs) and inflammatory cytokine (TNF-a). Lastly, rats were sacrificed and abdominal aorta was exposed for assessing MNV and RP using Doppler ultrasound.Results: In aged sedentary group, there were significant increase in SBP, RP, serum (TC, LDL-C, HDL-C) and serum TNF-a associated with significant decrement in MNV when compared to the corresponding values in the young control ones. Exercise training for six weeks significantly restored age-related vascular and biochemical changes except for HDL-C and TC there was insignificant diffrence when compared to the corresponding values in aged sedentary group.Conclusion: Regular exercise training in aging has a beneficial effect on the cardiovascular functions by accelerating blood flow and decreasing resistance to blood flow, also it has an additive beneficial effect on the associated dyslipidemia and chronic inflammatory state in aging.

Concurrent exercise improves insulin resistance and nonalcoholic fatty liver disease by upregulating PPAR-\u03b3 and genes involved in the beta-oxidation of fatty acids in ApoE-KO mice fed a high-fat diet

ObjectiveTo emphasize the mechanism of concurrent exercise effect on lipid disorders in insulin resistance (IR) and nonalcoholic fatty liver disease (NAFLD).Materials and methodsTwenty male ApoE knockout mice were randomly divided into two groups: HFD group (n = 10) fed a high fat diet, and HFDE group (n = 10) with high-fat diet intervention for 12 weeks and swimming exercise. Other ten healthy male C57BL/6 J mice were fed a normal diet, and included as control group. Retro-orbital blood samples were collected for biochemical analysis. Oil red O staining of liver tissues was performed to confirm the exercise effect. Western blotting was performed to evaluate the expressions of PPAR-γ, CPT-1, MCAD.ResultsThe levels of TG, TC, LDL, FFA, FIN, FPG and Homa-IRI in the HFD group were significantly higher than ND group, while these were markedly decreased in the HFDE group compared with HFD group. The Oil Red O staining of liver samples further confirmed the exercise effect on the change of lipid deposition in the liver. Western blotting showed increased expressions of PPAR-γ, CPT-1, MCAD induced by high fat diet were significantly downregulated by exercise.ConclusionA concurrent 12-week exercise protocol alleviated the lipid metabolism disorders of IR and NAFLD, probably via PPAR-γ/CPT-1/MCAD signaling.

Role of Protease Exosites for In Vivo Hemostatic Activity and Clearance of FIX

Introduction:FIX(a) binds rapidly and reversibly to vascular endothelium and extravascular matrix, in part mediated by the interaction of specific Gla domain residues with collagen IV. Both anticoagulant heparan sulphate and collagen IV localize predominantly to the basement membrane and supramolecular structure analysis suggests that heparan sulphate chains are integrated into collagen IV-containing networks. Previous work has demonstrated the contribution of the heparin and antithrombin (AT)-binding exosites in the protease domain to the regulation of human FIXa. Here we evaluate the contribution of these protease exosites to FIX hemostatic function and clearance in the hemophilia B (HB) mouse.Methods:Recombinant human FIX variants expressed in HEK293 cells with substitutions in the heparin- (K126A/K132A or R170A) or AT-binding (R150A) exosites (chymotrypsinogen #) were purified to homogeneity. In vivohemostasis was assessed in a saphenous vein bleeding model in HB mice. Anesthetized adult mice were injected with a weight-based dose predicted to achieve 30% FIX plasma concentrations and the exposed left saphenous vein was punctured with a 29 gauge needle. Clot formation at the injury site was repeatedly disrupted over 30 min to determine the average time to clot formation and total #clots/30 min. FIX pharmacokinetics (PK) in HB mice were determined with a weight-based dose injected into the tail vein. Serial retro-orbital blood samples were collected, plasma isolated and FIX content determined with a human FIX specific ELISA. FIX concentrations were plotted versus time and initially fit to the equation: [IX]t = [IX]max*(C1*e(-k1*t)+ C2*e(-k2*t)), where k1 and k2 are rate constants for the initial and terminal elimination phases, respectively. As the 4-parameter fit resulted in some large error estimates, the two phases were fit separately to estimate the respective parameters.Results:Previous characterization of APTT-based coagulant activities for these FIX variants (% WT ± SEM) were as follows: FIX WT 100 ± 1.4, R150A 73.6 ± 1.4, K126A/K132A 20.6 ± 9.2, and R170A 609.2 ± 37. In the saphenous vein bleeding assay, HB mice had no clot formation over 30 min, while wild type mice demonstrated 11 clots/30 min. When HB mice were injected with increasing concentrations of FIX WT (20%-100%), a dose dependent increase was observed from 4.3 to 16.5 clots/30 min (Table 1). When injected with sufficient FIX to achieve 30% plasma levels, the AT-binding exosite variant FIX R150A and heparin binding site variant K126A/K132A demonstrated similar hemostatic activity to FIX WT in the HB mouse. FIX R170A (30%) trended towards enhanced activity relative to WT, while 10% FIX R170A levels demonstrated similar activity to 30% FIX WT (not shown).The PK (5 min to 25 hr) of FIX was examined in HB and wild type mice. FIX WT demonstrated approximately ~30% recovery (initial peak) of predicted levels. FIX K126A/K132A had markedly enhanced recovery (2.2-fold higher than FIX WT), while FIX R150A and R170A demonstrated similar recovery to FIX WT (Table 1, Figure 1). FIX K126A/K132A demonstrated ~70% of predicted recovery. A rapid decrease in FIX levels was observed over the ensuing 4-6 hours with similar k1 values for all proteins. In contrast, terminal phase k2 values trended lower and predicted half-lives longer for the heparin-binding exosite mutants (FIX K126A/K132A and R170A). Consistent with occupation of extravascular sites by endogenous FIX, PK studies in wild type mice demonstrated ~20% enhancement of FIX recovery relative to HB mice (not shown).Conclusions:In contrast to in vitrocoagulant and thrombin generation activity, FIX K126A/K132A demonstrated similar in vivohemostatic activity to FIX WT, markedly enhanced recovery and modestly slower terminal clearance. The enhanced in vivo hemostatic activity of this variant relative to coagulant activity likely results from the markedly enhanced plasma FIX recovery. In contrast, FIX R150 demonstrates similar in vivo hemostatic activity and zymogen clearance to FIX WT. FIX R170A demonstrated ~3-fold enhanced hemostatic potency and similar zymogen clearance compared to FIX WT. This hemostatic potency is consistent with the enhanced coagulant activity of this FIX Padua (R170L) related variant. In addition to the collagen IV binding site, the FIX(a) heparin binding exosite is likely to play a substantial role in FIX localization to the extravascular compartment. [Display omitted] DisclosuresSheehan:Pfizer: Research Funding; Bayer: Consultancy, Research Funding.

Effect of Valsartan and Folic Acid on Vascular Reactivity, Leptin Hormone and Some Biochemical Parameters in Fat Diet-Induced Obese Male Rats

AbstractBackground: The development of a chronic low-grade inflammatory state has been shown to play a central role in the development of metabolic complications associated with obesity.Aim of Study: The aim was to determine the impact of oral administration of valsartan and folic acid to fat diet-induced obese male rats on the vascular reactivity & on the serum levels of leptin and some inflammatory and oxidative stress markers.Material and Methods: Fifty male albino rats of local strain were randomized into five equal groups: Control, obese, valsartan-treated obese, folic acid-treated obese and combined valsartan and folic acid-treated obese groups. Obesity was induced by feeding high fat diet and treatment was done for indicated groups for 16 weeks. Thereafter, weight and length were measured, and rats were subjected for rat tail-measurement of Systolic Blood Pressure (SBP). Then, fasting retro-orbital blood samples were collected for measuring serum Total Cholesterol (TC), Triglycerides (TGs), Low Density Lipoprotein-Cholesterol (LDL-C), High Density Lipoprotein-Cholesterol (HDL-C), Malondialdehyde (MDA), C-Reactive Protein (CRP) and leptin levels, as well as serum Glutathione Peroxidase (GPx) and Superoxide Dismutase (SOD) activities, andurinary nitrite/nitrate (NOx) level. Rats were tested for Vascular Reactivity to Acetylcholine (VRACh) and sodium nitroprusside (VRSNP) by Doppler flowmeter. Lastly, rats were sacrificed and abdominal visceral fats were resected and weighed.Results: High fat diet resulted in marked impairment of all the measured parameters, except VRSNP. Treatment of obese rats with valsartan resulted in improvement of BMI, adipose tissue mass, TC, LDL, CRP, leptin, NOx, MDA, SBP and VRACh; however, total body weight, TGs, HDL-C and antioxidant enzyme activities were not affected. Folic acid administration to obese rats improves all serum lipid param-eters, increased GPx and SOD activities, and showed a better effect than valsartan on NOx, leptin andVRACh, but less effect on CRP. On the other hand, folic acid had no effect on the anthropometric parameters and SBP of obese rats. Com-bined treatment showed a reduction in total body weight, and a significant better improvement of all the measured parame-ters, except MDA, than those of isolated valsartan or folic acid treatment, with normalization of MBI, TGs, LDL-C, NOx and VRACh.Conclusion: Both valsartan and folic acid, when admin-istered separately, ameliorate the detrimental effects of fat diet-induced obesity in rats. Combined valsartan and folic acid prophylactic treatment of fat diet-induced obese rats has an additive beneficial effect on the associated dyslipidemia, inflammatory state, oxidative stress, leptin hormone level, and endothelial dysfunction that is possibly achieved by different mechanisms of action.

Effect of Diabetes Mellitus and Its Control on Myocardial Contractile Function in Rats

AIM: This work was done to study the effect of both types of diabetes mellitus (DM) on myocardial contractility in rats. Also, we investigated the role of treatment of DM with insulin and rosiglitazone (used as treatment for type 1 and type 2 DM respectively) in improvement of myocardial dysfunction in diabetic rats.METHODS: The study included 50 male Wistar albino rats, divided into 5 groups: control (group I), streptozotocin induced type 1 DM (group II), fructose induced type 2 DM (group III), insulin treated type 1 diabetic rats (group IV) and rosiglitazone treated type 2 diabetic rats (group V). At the end of the study, retro-orbital blood samples were withdrawn and blood glucose, plasma triglyceride (TG), total cholesterol (TC) and thyroid hormones levels were measured. Rats were then anesthetized and their hearts were excised and connected to Langendorff apparatus to perform mechanical cardiac performance tests including heart rate (HR), left ventricular developed pressure (LVDP) and maximum rate of pressure rise (+dp/dt).RESULTS: Data of the study showed that relative to control group, there was significant increase in blood glucose, plasma TG and TC levels while, thyroid hormones and myocardial performance parameters showed significant decrease in both type 1 and type 2 diabetic rats. Treatment of type 1 diabetic rats with insulin and type 2 with rosiglitazone resulted in significant decrease in blood glucose, plasma TG and TC levels associated with significant improvement in thyroid hormones and myocardial performance parameters. The results also showed that insulin treatment of type 1 was more effective in ameliorating all parameters than treatment of type 2 by rosiglitazone.CONCLUSION: We concluded that the induction of both types of diabetes resulted in decreased myocardial performance parameters. The treatment of type 1 and type 2 diabetes by insulin and oral rosiglitazone respectively improved to a great extent the altered metabolism and mechanical myocardial parameters, with more improving effect of insulin in type 1 than rosiglitazone in type 2 DM.

QUERCETIN AMELIORATES DIABETIC NEPHROPATHY IN RATS VIA MODULATION OF RENAL NA<sup>+</sup>, K<sup>+</sup>-ATPASE EXPRESSION AND OXIDATIVE STRESS

Diabetic Nephropathy (DN) is one of most prevalent complications of Diabetes Mellitus (DM), therefore prevention of its development is a important field for researches. Quercetin is a plant flavenoid with hypoglycemic and antioxidant properties that is cla imed to have a reno-protective effect in diabetes. This study was designed to investigate the reno-preventi ve role of Quercetin treatment in terms of biochemi cal and pathological changes in diabetic rats and to de termine whether the effect is mediated through modulation of oxidative stress and Na + , K + ATPase expression and activity. Sixty male Sprague-Dawley rats were distributed equally among 6 groups: (i) Contro l group (C), (ii) Quercetin treated Control group ( CQ), (iii) Diabetic group (D), (iv) Diabetic Insulin tre ated group (DI), (v) Diabetic Quercetin treated gro up (DQ) and (vi) Diabetic Insulin and Quercetin treated gro up (DIQ). Systolic blood pressure was measured at t he end of the experiments (8 weeks). Retro-orbital blo od samples were used to determine the serum levels of glucose, HbA1c, urea, creatinine, Na + and K + . Renal homogenate levels of Na + , K + ATPase activity, Malondialdehyde (MDA0, Superoxide Dismutase (SOD) and Glutathione Peroxidase (GPx) were measured. Semiquantitative reverse transcriptase-PCR Na + , K + ATPase expression assays and kidney histopathological examination were conducted. Treatment with either insulin or Quercetin alone resulted in partial rever sal of the biochemical and histopathological signs of neph ropathy in diabetic rats. This was associated with partial but significant amelioration of indicators of oxida tive stress and Na + , K + ATPase gene expression and activity. However only combined treatment by both insulin and Quercetin significantly improved all of the aforementioned parameters up to the control levels. These results suggested that combined therapy with insulin and Quercetin might be a useful preventive tool aga inst development of DN.

Effect of experimental hyperthyroidism on the maximum acute exercise tolerance and neuromuscular performance in rats

Background Thyroid hormones regulate many physiologic functions such as energy and heat production, synthesis of proteins, and muscle contraction. Objective This study aims to demonstrate the mechanism by which hyperthyroidism affects neuromuscular performance and acute exercise tolerance and whether this effect is reversible or not after recovery in rats. Materials and methods Rats used in this study were divided into three groups, each containing 10 rats: (a) in the euthyroid group, rats were selected by measurement of serum FT3 (triiodothyronin), FT4 (tetraiodothyronine), and thyroid-stimulating hormone levels. (b) In the hyperthyroid group, hyperthyroidism was induced by Eltroxin in increasing doses, beginning from 50 μg to reach 200 μg/kg body weight daily by intragastric administration for 3 weeks. (c) In the recovery group, hyperthyroid rats are allowed to recover by exogenous thyroxine withdrawal for 25 days. After induction, body weight and the maximal swimming time were estimated, and then retro-orbital blood samples were collected for estimation of malondialdehyde and the total antioxidant capacity. Rats were then killed by cervical decapitation. Phrenic nerve diaphragm preparations were excised, connected to a four-channel oscillograph for measuring the strength of muscle contraction at the start of the experiment and after 30 min activity using both direct and indirect stimulation. Glucose uptake by the muscle during 30 min of activity and 30 min of recovery was estimated. Results The data show that hyperthyroidism induced by Eltroxin elevates serum malondialdehyde significantly when compared with the euthyroid group, whereas reduces total antioxidant capacity significantly. Hyperthyroidism was also associated with reduction of body weight, the maximal swimming time, the strength of skeletal muscle contraction induced by direct and indirect repetitive stimulation, and its glucose uptake. Conclusion Oxidative stress plays a critical role in the pathogenesis of hyperthyroidism, especially on skeletal muscle performance and exercise tolerance. This effect is reversible after restoration of the euthyroid state.

Additive effect of ozone therapy to insulin in the treatment of diabetic rats

Background Chronic hyperglycemia of diabetes is associated with long-term damage, dysfunction, and failure of various organs, especially the heart and blood vessels. Therapy in the past few decades was mainly aimed at reducing hyperglycemia. It became clear that ameliorating oxidative stress through treatment with antioxidants might be an effective strategy for reducing diabetic complications. Medical ozone treatment may be useful in the treatment of diabetes and its complications. Objective This study aims to show the additive effect of ozone to insulin in the treatment of diabetes in rats. Materials and methods Diabetes was induced by an intraperitoneal injection of streptozotocin (45 mg/kg) in 0.2 ml of 10 mmol/l citrate buffer. Rats were considered diabetic when fasting blood glucose was at least 113 mg/dl. Rats proved to be diabetic were isolated and subdivided into four subgroups: (a) diabetic nontreated rats (n = 8), (b) diabetic ozone-treated rats (n = 8), (c) diabetic insulin-treated rats (n = 8), and (d) diabetic insulin + ozone-treated rats (n = 8). After induction, all rats were fasted for 12 h. Systolic blood pressure (SBP) was measured. Retro-orbital blood samples were collected for estimation of fasting serum glucose, glycosylated hemoglobin, total antioxidant capacity (TAC), and malondialdehyde (MAD) level. Rats were then sacrificed; vascular reactivity to norepinephrine and acetylcholine with and without endothelial lining was estimated. Results The data showed that insulin reduced the elevated fasting serum glucose, glycosylated hemoglobin, MAD, and SBP significantly when compared with the diabetic nontreated group. Also, it significantly reduced TAC and vascular reactivity to norepinephrine with and without endothelium, but there was an increase in the percent of relaxation to acetylcholine. Ozone therapy potentiated the effects of insulin on SBP and vascular reactivity. Importantly, serum MAD and TAC and glycemic state were significantly improved. Conclusion This study shows that ozone therapy may have an additive effect in the treatment of diabetes by insulin; this may attribute to the multiprotective antioxidant effect of ozone.

Preventive roles of swimming exercise and pioglitazone treatment on hepatic dysfunction in a rat model of metabolic syndrome

Pioglitazone (Pio) and swimming exercise (SE) are insulin sensitisers. This investigation was suggested because of the significant side effects associated with Pio treatment in metabolic syndrome (MetS). This study was, therefore, designed to investigate the preventive role of Pio treatment and SE in terms of efficiency and pathological changes in MetS in a rat model. Sixty male Sprague-Dawley rats were distributed equally among 6 groups: (i) control group (C), (ii) exercised control group (C+E), (iii) Pio-treated control group (C+Pio), (iv) group with MetS, (v) group with MetS treated with Pio (MetS+Pio), and (vi) exercised MetS group (MetS+E). Systolic blood pressure and heart rate were measured at the end of the experiments (16 weeks). Retro-orbital blood samples were used to determine the serum levels of glucose, insulin, lipids, gamma glutamyl transferase, alanine transaminase, aspartate transaminase, alkaline phosphatase, fetuin-A, and adiponectin. Semiquantitative reverse transcriptase - PCR insulin gene expression assays and hepatic histopathological examination were conducted. Swimming exercise significantly improved all of the aforementioned parameters, more so than the Pio treatment. In particular, the serum hepatic enzyme levels and hepatic histopathological changes were improved compared with the MetS group. These results suggested that swimming exercise might be an alternative physiological preventive tool against hepatic dysfunction to avoid the side effects associated with Pio treatment, and this could be demonstrated in a rat model of metabolic syndrome.

Lack of P-selectin glycoprotein ligand-1 protects mice from thrombosis after collagen/epinephrine challenge

IntroductionIn thrombotic processes, during the association of leukocytes with platelets and endothelial cells, P-selectin glycoprotein ligand-1 (PSGL-1) binds to P-selectin, expressedon activated platelets and endothelial cells. Our aim was to establish the role of PSGL-1 in thrombus formation by evaluating the response to thrombotic stimuli in wild type and PSGL-1 knockout mice. Materials and methodsMice were challenged by tail vein injection of (i) 15μg collagen plus 3μg epinephrine (coll/epi) (ii) 7.5μg collagen plus 1.5μg epinephrine or (iii) saline. Retro-orbital blood samples were collected in ACD anticoagulaed tubes and platelet and leukocyte counts were measured. In addition, kidneys, liver, spleen and lungs were investigated for fibrin deposition by immunohistochemistry and Western-blotting. Frozen sections were analysed for double labeling for platelet and leukocyte presence. ResultsAfter coll/epi challenge, the number of platelets and leukocytes decreased significantly in both genotypes. Lower agonist concentration resulted in an attenuated platelet decrease in PSGL-1 knockout mice compared to the controls, however changes in leukocyte and neutrophil counts were not significantly different in the two strains. In knockout mice considerably less fibrin deposition has been observedin the lungs by Western-blotting and immunohistochemistry. After coll/epi challenge the lungs of the PSGL-1 knockout animals contained both platelets and leukocytes but less thrombi has been detected than in wild-type mice. ConclusionsOur results indicate that the deficiency of PSGL-1 results in milder thrombocytopenia, less fibrin deposition and lower number of thrombosed blood vessels, suggesting that this molecule is essential for multicellular interactions during thrombus formation.

Effect of vanadium on renal Na+,K+-ATPase activity in diabetic rats: a possible role of leptin

Several researches attempt to protect diabetic patients from the development of nephropathy. Involvement of leptin and renal Na+,K+-ATPase enzyme in diabetic nephropathy (DN) development is a recent field for researches. Vanadium, as a trace element with insulin mimetic effect, may act synergistically with insulin to protect against the development of DN. Sixty male Sprague Dawley rats were divided into six groups: control group (C), vanadium control group (CV), streptozotocin-induced diabetic group (D), insulin-treated diabetic group (DI), vanadium-treated diabetic group (DV), and combined insulin and vanadium-treated diabetic group. Six weeks later, systolic blood pressure (SBP) was measured and retro-orbital blood samples were collected to estimate glycosylated hemoglobin (HbA(₁c)), serum sodium (Na+) and creatinine, blood urea nitrogen (BUN) and plasma leptin levels. Preparation of microsomal fraction of renal tissue homogenate for estimation of Na+,K+-ATPase activity was done. The D group showed a significant increase in SBP, HbA(₁c), serum Na+, creatinine, and BUN levels and Na+,K+-ATPase activity in microsomal fraction of renal tissue homogenate while plasma leptin level decreased significantly compared with C and CV groups. Both DI and DV groups showed a significant improvement in all the above measured parameters compared with D group while there were no significant changes between the DI and DV groups. Concomitant treatment with insulin and vanadium resulted in a significant improvement in all the measured parameters compared to each alone. Vanadium in combination with insulin ameliorates DN markers and reduces renal Na+,K+-ATPase overactivity in diabetic rats. An effect that may be partially mediated through correction of hypoleptinemia observed in these animals.

A JAK2 Interdomain Linker Relays Epo Receptor Engagement Signals to Kinase Activation

JAK2 (Janus kinase 2) is essential for cytokine receptor signaling, and several lines of evidence support a causal role of an activating JAK2 mutation in myeloproliferative disorders. JAK2 activity is autoinhibited by its pseudokinase domain in the basal state, and the inhibition is released by cytokine stimulation; how engagement of the cognate receptor triggers this release is unknown. From a functional screen for gain-of-function JAK2 mutations, we discovered 13 missense mutations, nine in the pseudokinase domain and four in the Src homology 2 (SH2)-pseudokinase domain linker. These mutations identified determinants for autoinhibition and inducible activation in JAK2. Two of the mutants, K539I and N622I, resulted in erythrocytosis in mice. Scanning mutagenesis of the SH2-pseudokinase domain linker indicated that its N-terminal part was essential for interaction of JAK2 with the Epo receptor, whereas certain mutations in the C-terminal region conferred constitutive activation. We further showed that substitutions for Glu(543)-Asp(544) in this linker or Leu(611), Arg(683), or Phe(694) in the hinge proximal region of the pseudokinase domain resulted in activated JAK2 mutants that could not be further stimulated by Epo. These results suggest that the SH2-pseudokinase domain linker acts as a switch that relays cytokine engagement to JAK2 activation by flexing the pseudokinase domain hinge.