Cilia Have a Significant Role in Regulating Cell Size in Response to Fluid Flow Induced Shear Stress in a Flow Chamber

Cilia are hair-like protrusions on the apical surface of cells. Their function is to relay mechanical signals like shear stress from extracellular into intracellular environment and thereby maintain cellular homeostasis. Ciliary dysfunctions include polycystic kidney disease and new therapeutic interventions based on ciliary function are under investigation. The current study evaluates the use of a custom designed fluid flow chamber’s ability to study the role of cilia in regulating cell size in response to shear stress. A fluid flow chamber that continually maintains laminar flow at different flow rates and temperature was designed. Endothelial wild type cells (ETWT) that have cilia and polycystic kidney disease cells (PKD) that lost their ciliary function are grown on different glass slides. Cells on each glass slide are then exposed to continuous flow of phosphate-buffered saline at 37oC in the flow chamber. The optimal flow rate and duration of flow were first determined by measuring the total protein concentration before and after exposing the cells. Cell radius and area before and after exposing them to flow are measured using the NIS Software available on the microscope. The results from protein concentrations (n=12) indicate that cells are still attached at normal physiological flow rate 467 mL/min (2.8 µg/µL) and did not significantly differ from 60 mL/min (4.08 µg/µL) or 600 mL/min (2.73 µg/µL). The results for duration of fluid flow (n=22) show that 60 minutes (0.09 + 0.01 µg/µL) is optimal compared to 120 minutes (0.06 + 0.01 µg/µL) or 180 minutes (0.10 + 0.02 µg/µL). Under these optimal conditions, the average area of ETWT cells (n=300) measured from different slides before and after the flow is 4420.81+ 67.40 µm2 and 4678.17 + 87.15 µm2 (n=200) respectively. For PKD cells, the average area before and after the flow (n=300) is 5682.46 + 105.48 µm2 and 4173.74 + 263.97 µm2 (n=250). These results are in agreement with the published literature on the ability of cilia to maintain cell size in ETWT cells in response to shear stress that is similar to normal blood flow. However, under similar conditions, PKD cells could not maintain their cell size as the mechano-chemical signaling pathway that communicates external signals to prepare appropriate intracellular response is disrupted. These results provide confirmation that the custom designed parallel plate fluid flow chamber is a reliable tool to investigate the specific targets in the mechano-chemical cell signaling pathways

Effects of Physical Stress and Maturational Changes on Hypothalamic Pituitary Adrenal Axis Function Through Cortisol Analysis

Cortisol is a versatile hormone that possesses both catabolic and anabolic functions in the body, such as increasing the blood glucose levels through gluconeogenesis and metabolizing carbohydrates, proteins, and fats.1234 Cortisol levels are controlled by communication of the hyptothalamic-pituitary-adrenal axix (HPA axis). Plasma concentration of cortisol adheres to a diurnal rhythm, meaning that cortisol levels are highest in the morning and taper off throughout the day

Evaluating the Bioavailability of Carbamazepine Using a Novel SNEDDS Formulation

Carbamazepine (CBZ) is an anticonvulsant drug primarily used to treat epilepsy, bipolar disorder, trigeminal and glossopharyngeal neuralgia. CBZ is a lipophilic, poorly soluble drug that belongs to the class-2 category according to the Biopharmaceutics Classification System. As a class-2 drug, the plasma concentration of CBZ is limited by its ability to diffuse across biological membranes. To increase its bioavailability, different methods such as crystal modifications, particle size reduction, amorphization, cyclodextrin complexation, pH modification, and self-emulsification were explored. Of these methods, Self Nano Emulsifying Drug Delivery Systems (SNEDDS) have shown to reduce particle size of CBZ molecules and improve its solubility. However, the bioavailability of CBZ administered as SNEDDS are not yet investigated. Given this background, the current study proposes to evaluate the bioavailability of these novel drug delivery systems using a rat model. The study is designed as a randomized controlled crossover experiment using 10-12 Sprague-Dawley rats divided equally into two groups. For this study, blood samples will be collected at 5, 10, 15, 20, 30, 45, 60, 90, and 120 minutes after administering two different formulations of CBZ nanoemulsions and stored at -20°C until ready for analysis. Plasma concentrations of CBZ will be determined by HPLC method. An unpaired t-test will be used to compare the significance between the two sets of data

Evaluating the Bioavailability of Carbamazepine Using a Novel SNEDDS Formulation

Central to the mechanism of how drugs work are the concepts of solubility and bioavailability. Drugs enter the body via absorption into the bloodstream, arrive at the target location, and bind to receptors to cause an effect. Drugs need to be soluble enough to pass through the cell membrane to enter and exit the bloodstream. Higher solubility generally correlates to higher bioavailability. Additionally, the smaller the particle size, the easier the drug will pass through the membrane into the blood plasma. Researchers have designed a system to categorize solubility class: Class I being high permeability and high solubility, Class II high permeability and low solubility, Class III low permeability and high solubility, and Class IV low permeability and low solubility. The study will use a Class II anticonvulsant, carbamazepine (CBZ). CBZ is a suitable candidate for this study because it requires a higher bioavailability due to its need to cross the blood brain barrier and act on the trigeminal nucleus. To increase bioavailability researchers have tried crystal modifications, particle size reduction, amorphization, cyclodextrin complexation, pH modification, and self-emulsification. These methods have been successful at increasing bioavailability, but this experiment will focus on reducing particle size into a new self-emulsifying formulation. In particular, the formulation of CBZ in this study is a self nano-emulsifying drug delivery system (SNEDDS), which shows more promise than previous methods to increase bioavailability. This study will create a SNEDDS formulation as a nasal nebulizer mist delivery and compare it to a FDA approved oral suspension using a crossover rat model design. Sixteen Sprague-Dawley rats will be ordered through Central State University and normalized to the study environment for a minimum of one week. Pending IACUC approval from Central State University, the tail vein method will be used to collect blood samples. The samples will be stored until needed for analysis using ELISA, enzyme-linked immunosorbent assay, which will be used to determine the concentration of CBZ in blood plasma

The Effect of Andrographolide on the Metabolism of Carbamazepine in Rats

Objective: To determine if andrographolide (AND) impacts the pharmacokinetics of carbamazepine (CBZ). Background: CBZ is an anticonvulsant medication that is metabolized in the liver by cytochrome P450 (CYP) enzymes. AND is an over-the-counter medication that is common in Eastern cultures to treat inflammation and is a CYP enzyme inhibitor. Because CBZ is metabolized in the liver by these specific CYP enzymes, coadministration of andrographolide and CBZ could result in a herb-drug interaction. Methods: Sprague-Dawley rats (N=12) aged between 3 months and 6 months (250-350 g) will be split into control (N=6) and treatment (N=6) groups. The treatment group will receive an AND injection (dissolved in dimethyl sulfoxide to 10 mg/mL) intraperitoneally for seven consecutive days. On the eighth day, another AND injection will be administered intraperitoneally as well as an injection of CBZ (CBZ powder prepared into a 20 mg/mL emulsion) administered via intravenous route. Plasma samples will be collected every 20 minutes for 4 hours and stored at -20℃. Analysis: HPLC analysis will yield a time vs. plasma concentration graph that will allow us to calculate the rate of elimination (K). The mean K value will be determined for both the control and treatment group. The mean K value of 6 rats in the treatment group will be compared to the mean value of K of the 6 rats in the control group. These will be analyzed using SPSS and utilizing an unpaired t test, with a p\u3c0.05 deemed statistically significant

Herb-Drug Interaction of Andrographolide on the Pharmacokinetics of Carbamazepine in Rats

Objective: To determine if andrographolide (AND) impacts the pharmacokinetics of carbamazepine (CBZ). Background: CBZ is an anticonvulsant medication that is metabolized in the liver by cytochrome P450 (CYP) enzymes. AND is an over-the-counter medication that is common in Eastern cultures to treat inflammation and is a CYP enzyme inhibitor. Because CBZ is metabolized in the liver by these specific CYP enzymes, coadministration of andrographolide and CBZ could result in a herb-drug interaction. Methods: Sprague-Dawley rats (N=12) aged between 3 months and 6 months (250-350 g) will be split into control (N=6) and treatment (N=6) groups. The treatment group will receive an AND injection (dissolved in dimethyl sulfoxide to 10 mg/mL) intraperitoneally for seven consecutive days. On the eighth day, another AND injection will be administered intraperitoneally as well as an injection of CBZ (CBZ powder prepared into a 20 mg/mL emulsion) administered via intravenous route. Plasma samples will be collected every 20 minutes for 4 hours and stored at -20℃. Analysis: HPLC analysis will yield a time vs. plasma concentration graph that will allow us to calculate the rate of elimination (K). The mean K value will be determined for both the control and treatment group. The mean K value of 6 rats in the treatment group will be compared to the mean value of K of the 6 rats in the control group. These will be analyzed using SPSS and utilizing an unpaired t test, with a

Maturational Changes in Myosin Light Chain Kinase Activity in Ovine Carotids

Vascular reactivity changes dramatically during postnatal maturation due in large part to developmental changes in myofilament calcium sensitivity. Recent findings suggest that reactivity of the thick filament component of calcium sensitivity is upregulated in fetal compared to adult arteries. In light of these findings, the present study tests the hypothesis that upregulation of fetal thick filament reactivity is due to upregulation of myosin light chain kinase (MLCK) activity. To test this hypothesis, MLCK abundance and its activity is measured in intact arteries. The results indicate that MLCK abundance is 6.03 ± 0.96 fold greater in adult than in fetal arteries. Total MLCK activity (%MLC phosphorylated/sec) estimated as the rate of phosphorylation of myosin light chain in intact arteries was greater in fetal (7.39 ± 0.53) than in adult (6.56 ± 0.29) arteries. When total MLCK activity was normalized relative to MLCK & MLC abundance to estimate the apparent specific activity of MLCK (ng MLC phosphorylation/sec/ng MLCK), these estimates were dramatically greater in fetal (1.52 ± 1.11) than in adult (0.26 ± 0.01) arteries. Further, to test if these differences are due to differences in fractional activation of the enzyme, maximum velocity (Vmax) of MLCK was estimated in artery homogenates. The results indicate that Vmax (ng MLC phosphorylated/sec) is significantly greater in fetal (163 ± 11) compared to adult (130 ± 9) arteries. When fractional activation was calculated the results showed about 4.9 ± 0.3 fold greater activation of MLCK in fetal compared to adult arteries. Together, these results support the hypothesis that upregulation of fetal thick filament reactivity is due to upregulation of MLCK activity. These studies were the first to offer a quantitative assessment of age related differences in MLCK activity in intact arteries and indicate the relative extents to which changes in MLCK abundance, activity and fractional activation contribute to these differences. From a clinical science perspective, these studies help in understanding the mechanisms involved in adaptation of fetal vascular system for postnatal life so that, new strategies of pharmacological management of NICU neonates with cerebrovascular and cardiovascular instabilities can be developed