Iontophoretic transdermal delivery of glycyrrhizin: effects of pH, drug concentration, co-ions, current intensity, and chemical enhancers.

The aim of the present study was to evaluate the feasibility of transdermal delivery of glycyrrhizin, an agent used in the treatment of chronic hepatitis C, by cathodal iontophoresis using Ag/AgCl electrodes in vitro. The effects of donor pH (pH 4-7), concentration of drug (0.025-0.2% (w/v)), concentration of external chloride ions (Cl(-)) (0-133 mM), current strength (0-0.5 mA/cm(2)), and permeation enhancers (urea and Tween 80) on the skin permeability of glycyrrhizin were examined in in vitro skin permeation studies using porcine ear skin as the membrane. The cumulative amount of permeated glycyrrhizin and the steady-state skin permeation flux of glycyrrhizin across porcine skin increased in a pH-dependent manner. The skin permeability of glycyrrhizin was independent of the concentration of drug and competed only with a high external Cl(-) concentration. The skin permeation flux of glycyrrhizin increased with the current (R(2)=0.8955). The combination of iontophoresis and enhancers provided an additive or synergistic effect, and a skin permeation flux of about 60 µg/h/cm(2) was achieved. The plasma concentration of glycyrrhizin in humans, extrapolated from the in vitro steady-state permeation flux across porcine skin, was within the therapeutic level. These results suggest that cathodal iontophoresis can be used as a transdermal drug delivery system for glycyrrhizin using reasonable patch sizes and acceptable levels of current intensity.

Pharmacologically distinctive behaviors other than burying marbles during the marble burying test in mice.

In the marble burying test, we focused on the 5 distinctive behavioral parameters of mice other than burying marbles, i.e. digging, latency to the first digging, exploration around marbles, rearing and locomotor activity. Typical anxiolytics or antidepressants with different mechanisms, fluvoxamine (30 mg/kg, selective serotonin reuptake inhibitor), bupropion (60 mg/kg, noradrenaline and dopamine reuptake inhibitor), imipramine (60 mg/kg, tricyclic antidepressant) and diazepam (10 mg/kg, benzodiazepine) were used to examine whether these behavioral parameters are sensitive to pharmacological treatments. Each of the drugs demonstrated an individual action pattern on the 4 behavioral parameters (latency to the first digging, exploration around marbles, rearing and locomotor activity). On the other hand, all 4 drugs reduced burying marbles and digging, which were correlated with each other. These results suggest that the former 4 behavioral parameters are sensitive to pharmacological treatment and that pharmacological regulation mechanisms of them may be different from burying marbles and digging. They could be useful to identify the type of action of a test drug like selective serotonin reuptake inhibitor, noradrenaline and dopamine reuptake inhibitor, tricyclic antidepressant or benzodiazepine.

Neurochemical responses to antidepressants in the prefrontal cortex of mice and their efficacy in preclinical models of anxiety-like and depression-like behavior: a comparative and correlational study.

RATIONALE: Marble burying and forced swimming behavior are widely used and sensitive tests for identifying clinically effective antidepressant drugs, although the underlying neurobiology of these behaviors is not fully elucidated. OBJECTIVES: The objective of this study was to determine the relationship between the behavioral effects of antidepressant drugs and their ability to modulate extracellular neurotransmitter levels in the prefrontal cortex. MATERIALS AND METHODS: The effects of fluoxetine, fluvoxamine, citalopram, imipramine, and desipramine (0 to 60 mg/kg by oral gavage, except fluoxetine at 0 to 40 mg/kg) were studied independently in CD-1 mice in the marble-burying task, forced swim task and on extracellular concentrations of serotonin, norepinephrine, and dopamine in the prefrontal cortex by freely moving microdialysis. RESULTS: Fluvoxamine, fluoxetine, and citalopram all suppressed marble-burying behavior, but produced no change in immobility time in the forced swim test. In contrast, imipramine and desipramine suppressed both marble-burying behavior and increased swimming time in the forced swim test, although desipramine mildly suppressed locomotor activity at the maximal dose. Fluvoxamine, fluoxetine, and citalopram all increased extracellular levels of cortical serotonin. Desipramine and imipramine increased extracellular dopamine levels. Fluoxetine, desipramine, and imipramine increased extracellular norepinephrine levels. Correlational analysis revealed a positive correlation between efficacy of drugs in the forced swim test and cortical extracellular dopamine levels, whereas a positive correlation was found between efficacy in the marble-burying test and extracellular serotonin levels. CONCLUSIONS: Although marble burying and forced swimming behavior have strong predictive validity in tests of antidepressant action, each assay appears to be underpinned by entirely different neurochemical systems.

A new method for producing urinary bladder hyperactivity using a non-invasive transient intravesical infusion of acetic acid in conscious rats.

INTRODUCTION: Animal models that closely resemble the pathophysiology of human overactive bladder are important for evaluating novel therapeutics to treat the disorder. We established a non-invasive hyperactive bladder model that is sensitive to anti-muscarinic drugs and without bladder inflammation. METHODS: Acetic acid solution was infused into the bladder for 5 min via the urethral orifice without any surgical procedures under isoflurane anaesthesia. After washing the bladder with saline, voiding frequency (VF) and total urine volume were determined for 9 h under conscious conditions. RESULTS: Infusion of a 0.5% acetic acid solution caused a significant increase in VF, without influencing total urine volume or inducing significant histopathological inflammatory alterations in the bladder urothelium. Oral administration of oxybutynin (3 and 10 mg/kg) significantly ameliorated increases in VF induced by 0.5% acetic acid. Infusion of 0.75% acetic acid induced intensive urinary inflammation and a decrease in total urine volume as well as an increase in VF. Oral treatment with oxybutynin (10 mg/kg) did not significantly improve the increased VF due to 0.75% acetic acid. Acetic acid (0.5%) infusion evoked bladder hyper-responsiveness whether applied at night or during the day. However, VF was increased more by the nighttime application of acetic acid, while there were no significant differences in basal levels of VF between daytime and nighttime. DISCUSSION: In this study, the non-invasive rat urinary hyperactive bladder model indicated minimizes the secondary effects of experimental procedures such as surgical operations and anesthesia on bladder function and is sensitive to oxybutynin. Thus, the model may be useful for investigating novel therapeutics for OAB treatment.

Inhibition of phosphodiesterase type 4 decreases stress-induced defecation in rats and mice.

BACKGROUND/AIMS: Phosphodiesterase type 4 (PDE4) has been previously shown to regulate colonic contractile activity in vitro. In this study, the effects of PDE4 inhibition were assessed in a model of stress-induced defecation previously demonstrated to be due to increased colonic transit/evacuation. METHODS: Rats were individually placed in a mild restraint cage and placed into a 12 degrees C environment (cold-restraint stress) for 60 min. Mice received restraint (only) stress at room temperature for 30 min. Loperamide (positive control compound) or two different PDE4 inhibitors (rolipram and roflumilast) were administered orally at several doses to the rodents 1 h before stress began. Vehicle alone was administered for comparison. The number of fecal pellets expelled during stress (fecal pellet output), total fecal pellet wet weight and total fecal water content were measured. RESULTS: Loperamide produced a dose-related decrease (ID(50)s in mg/kg) in fecal pellet output (rat = 7.4, mouse = 0.7) and significantly decreased fecal wet weight (72.9%) and decreased fecal percent water content (9.4%). The two PDE4 inhibitors produced a similar dose-related inhibition of fecal pellet output. Rolipram exhibited ID(50)s in rat and mouse of 14.1 and 27.1, respectively. Rolipram significantly decreased fecal wet weight (58.8%) but increased fecal percent water content (15.0%). For roflumilast, ID(50)s were 24.2 mg/kg and 12.4 in the rat and mouse, respectively. Although roflumilast also significantly (p < 0.05) decreased fecal wet weight (47.2%), it did not significantly increase fecal percent water content. CONCLUSIONS: These data indicate that PDE4 inhibition is effective in reducing rodent stress-induced defecation, provides the first functional data on a potential role for PDE4 activity in the colonic evacuation response to stress, and indicates the potential utility of PDE4 inhibitors in functional bowel disease such as irritable bowel syndrome requires further evaluation.

Immunolocalization of farnesoid X receptor (FXR) in mouse tissues using tissue microarray.

Farnesoid X receptor (FXR) is a member of the nuclear receptor family and is known to play important roles in bile acid homeostasis, and lipid and glucose metabolism. In this study, to elucidate the systemic physiological functions of FXR, comprehensive immunohistochemical analysis of cell/subcellular localization of FXR and its heterodimer partner, retinoid X receptor (RXR)-alpha, in adult mice tissues was performed using tissue microarray (TMA)-based immunohistochemistry. FXR immunolabeling was observed in the enterohepatic system--including absorptive epithelium in the intestines, hepatocytes and gall bladder epithelium, several epithelial lineage cells including the basal cells of stratified epithelium in the tongue, esophagus, forestomach--skin, corneal epithelium and ciliary body epithelium in the eye and adrenocortical cells--including glandular cells in the zona reticularis/fasciculata. In these FXP-positive cells, FXR was preferentially localized to the nucleus. RXR-alpha was ubiquitously distributed in the nucleus of most cell types, including FXR-positive cell types in the examined tissues. These data suggest that FXR might have various physiological roles, not only in bile acid homeostasis, and lipid and glucose metabolism, but also in the epithelial cell barrier, visual and urinary function through multiple organ systems.

Changes in micro-CT 3D bone parameters reflect effects of a potent cathepsin K inhibitor (SB-553484) on bone resorption and cortical bone formation in ovariectomized mice.

Cathepsin K is a cysteine proteinase that is highly expressed by osteoclasts and is being pursued as a potential drug target for the treatment of osteoporosis. We have reported that microcomputed tomography (micro-CT) analysis of bone microarchitecture may serve as a valuable tool for evaluating both antiresorptive and anabolic agents in ovariectomized (OVX) mice. The purpose of this study was to evaluate the effect of SB-553484, a novel cathepsin K inhibitor (human Ki,app=0.14 nM, mouse Ki,app=26 nM), on the OVX mice by micro-CT bone morphometric analysis. Seven weeks female BALB/c mice were OVX or sham-operated. OVX animals were treated with SB-553484 (30 mg/kg, sc) or Rolipram (10 mg/kg, po), a phosphodiesterase 4 inhibitor used as a positive bone anabolic agent, twice a day for 2 weeks. Both SB-553484 and Rolipram significantly prevented the decrease of trabecular bone volume as well as the deterioration of trabecular architecture in OVX mice. Interestingly, SB-553484 demonstrated a more pronounced effect in improvement of trabecular separation, number and connectivity, and a weaker effect in improvement of trabecular thickness compared to that of Rolipram. These differences indicate that SB-553484 mainly acted as an antiresorptive agent in OVX-induced loss of trabecular bone. On the other hand, SB-553484 significantly increased cortical bone volume and cortical thickness as well as Rolipram in OVX mice indicating an unexpected stimulatory effect of SB-553484 on cortical bone formation. These data suggest that targeting cathepsin K may prove therapeutically beneficial in the treatment of diseases with accelerated bone loss such as postmenopausal osteoporosis not only by inhibiting bone resorption but also by potentially stimulating cortical bone formation.

Receptor-activated Smad localisation in bleomycin-induced pulmonary fibrosis.

BACKGROUND: Recent advances in fibrosis biology have identified transforming growth factor (TGF)-beta type I receptor-mediated activation of Smads as playing a central part in the development of fibrosis. However, to date, there have been few studies that examined the localisation and distribution of receptor-activated Smads protein (R-Smads: Smad2 and 3) during the fibrosis progression. AIMS: To histopathologically assess the time-course change of the localisation and distribution of the Smads protein in pulmonary fibrosis. METHODS: Pulmonary fibrosis was induced by intranasal injection of bleomycin (0.3 U/mouse). Lungs were isolated 2, 5, 7, 9 and 14 days after bleomycin treatment. Histological changes in the lungs were evaluated by haematoxylin-eosin stain or Masson's trichrome stain, and scored. TGF-beta1, Smad3 and phosphorylated Smad2 localisations in lung tissues were determined by immunohistochemistry. RESULTS: The bleomycin treatment led to considerable pulmonary fibrotic changes accompanied by marked increase in TGF-beta1 expression in infiltrating macrophages. With the progression in fibrosis (day 7-14), marked increases in Smad3-positive and pSmad2-positive cells were observed. There were intense Smad3-positive and pSmad2-positive signals localised to the nuclei of the infiltrating macrophages and to type II epithelial cells, and less intense signals in fibroblasts and hyperplastic alveolar/bronchiolar epithelial cells. CONCLUSIONS: The time-course data of TGF-beta1 and R-Smads indicate that progressive enhancement of TGF-beta1 signalling via R-Smad is activated in the process of fibrosis progression.

Expression profiling of liver receptor homologue 1 (LRH-1) in mouse tissues using tissue microarray.

Liver receptor homologue 1 (LRH-1) is a nuclear receptor that plays important roles in lipid homeostasis and embryogenesis. To elucidate systemic physiological functions of LRH-1, we used tissue microarray-based immunohistochemistry to examine the tissue distribution and localization of LRH-1 in adult mouse tissues. LRH-1 immunoreactivity was observed in the nucleus of multiple epithelial lineage cells in the digestive system (including absorptive epithelial cells in the small and large intestines, goblet cells, acinar cells of the exocrine glands, chief cells and mucus neck cells in the stomach, granular and prickle layer cells in the tongue and forestomach, and gall bladder epithelium); respiratory system (alveolar type II cells); and urinary system (transitional epithelium). Nuclear LRH-1 immunoreactivity was also localized in cells involved in fatty acid/glucose metabolism, including hepatocytes, brown adipocytes, and cardiomyocytes, and neurons involved in the regulation of food intake, including the arcuate nucleus in the hypothalamus and paraventricular nucleus of thalamus. Additionally, LRH-1 immunoreactivity was observed in testicular Leydig cells and ovarian follicular cells. These data suggest that LRH-1 functions in multiple organ systems to regulate epithelial cell physiology and differentiation, energy metabolism, and reproduction.

Kv7.2-7.5 voltage-gated potassium channel (KCNQ2-5) opener, retigabine, reduces capsaicin-induced visceral pain in mice.

K(v)7.2-7.5 voltage-gated potassium channels (KCNQ2-5) are associated with M-current and known to distribute in the nociceptive sensory pathway (e.g., dorsal root ganglia and spinal cord). Opening of these channels leads to cell membrane hyperpolarization that results in decreased neuronal action potentials. Since, KCNQ/M-current is located in the visceral sensory system, we examined the anti-nociceptive effect of the KCNQ opener, retigabine, on visceral pain induced by an intracolonic injection of capsaicin in mice. Intraperitoneal administration of retigabine (1, 3 and 10 mg/kg) dose-dependently suppressed visceral pain behavior (i.e., the number of licking) induced by the capsaicin treatment and prolonged the latency to first licking. These data provide the first evidence that increased KCNQ channel conductance plays an inhibitory role in the visceral pain pathway.

T cells are involved in the development of arthritis induced by anti-type II collagen antibody.

T cells play an important role in initiating autoimmune responses and maintaining synovial inflammation in rheumatoid arthritis. Although, anti-type II collagen antibody-induced arthritis (CAIA) is generally believed to be a T cell- and B cell-independent model, the detailed pathogenesis of CAIA remains unclear. In the present study, to elucidate the contribution of T cells to the pathogenesis of CAIA, we evaluated the effects of CTLA4 Ig and cyclosporin (CsA). Arthritis was induced in mice by intravenous injection of anti-type II collagen antibody followed by intraperitoneal injection of lipopolysaccharide. CTLA4 Ig was intraperitoneally administered and CsA was subcutaneously administered; then the severity of arthritis was evaluated by scoring the edema and erythema of paws and by measuring hind paw thickness. Paw samples were collected 12 days after the antibody injection, and the mRNA expression levels were analyzed by real-time quantitative polymerase chain reaction. Administration of CTLA4 Ig ameliorated the increases in arthritic score and paw thickness in the later phase, but not in the early phase of arthritis. CsA suppressed the increases in arthritic score and paw thickness in both the early and later phases of arthritis. CTLA4 Ig and CsA suppressed mRNA up-regulation of T-cell markers, CD3 and CD25, and immune response-related mediators, IFN-gamma and IL-12. They also suppressed the up-regulation of macrophage marker, F4/80, and proinflammatory cytokines, TNF-alpha, IL-1beta and IL-6. The results provide direct evidence that arthritis in this model is T-cell activation dependent.

A new automated and high-throughput system for analysis of the forced swim test in mice based on magnetic field changes.

INTRODUCTION: MicroAct (Neuroscience, Tokyo, Japan) was originally developed as a new system for an automated analysis of scratching behaviors in mice. This system is based on the detection of electric current in the coils according to the movement of the magnets implanted in the hind paws. We applied and improved this system to establish an automated analysis system of forced swimming behaviors in mice, which is used as an animal model of depression. METHODS: One day before the test, male CD-1 mice were attached with a wire ring to their hind paws under inhalation anesthesia. After attaching a small magnet to both of the wire rings, each animal was placed for 6 min in a glass cylinder filled with water, which is surrounded by a coil. The swimming behaviors of the mouse were analyzed for the measurement of duration of immobility, a major marker of depression in rodents, by the detection system (MicroAct). The duration of immobility was also determined by manual measurement using the swimming behavior-recorded videotapes produced at the same time as the automated analysis. RESULTS: The difference of the duration of immobility between naïve mice and mice with the rings was not significant. The dose-response effect of imipramine (tricyclic antidepressant, 0, 7.5, 15, 30 and 60 mg/kg, p.o.) on the duration of immobility in the last 4-min of the 6-min testing period determined by MicroAct was similar to that assessed by the manual measurement. These data from the two different methods were significantly correlated (r=0.8805). Moreover, throughput of the automated analysis was 15 times more efficient than that of the manual analysis. DISCUSSION: These results suggest that the automated analysis system of forced swimming of mice using MicroAct can be used as a high-throughput method to examine antidepressive activity of a compound with objectivity and reliability.

Analysis of change patterns of microcomputed tomography 3-dimensional bone parameters as a high-throughput tool to evaluate antiosteoporotic effects of agents at an early stage of ovariectomy-induced osteoporosis in mice.

OBJECTIVES: The purposes of this study were to develop an osteoporosis model in a short period of 2 weeks after ovariectomy in mice and to investigate whether analysis of microcomputed tomography (muCT) 3-dimensional bone parameters could provide useful information on the mechanism of action of antiosteoporotic agents. MATERIALS AND METHODS: Mice were ovariectomized (OVX) or sham-operated, and the OVX mice were treated daily with 17beta-estradiol (E2), parathyroid hormone (PTH[1-34]), raloxifene, rolipram, or vehicle for 2 weeks. On day 14 post-OVX, the left femur bones were removed and then the distal metaphyseal bone was analyzed by both muCT and histomorphometry. RESULTS: The trabecular bone volume, thickness, number, and connectivity significantly decreased and the number of osteoclasts increased in OVX mice. Treatment of OVX animals with each of the 4 antiosteoporotic agents significantly increased the bone volume and improved the bone architecture. However, the improvement of trabecular thickness in the rolipram-treated group and that of cortical thickness in the PTH(1-34)-treated group were the most marked, whereas the improvement of connectivity in the rolipram-treated group was the least among the drug-treated groups. These different improving effects of agents on the bone parameters reflect the differential effects of these agents on bone formation and bone resorption. CONCLUSIONS: This study demonstrated the feasibility of evaluating the effect of the antiosteoporotic agents within 2 weeks after ovariectomy in mice. The muCT analysis may serve as a valuable tool, specifically in a high-throughput pharmacological screening test, offering useful information regarding the effects of test compounds on both bone resorption and formation.

In vitro and in vivo transdermal iontophoretic delivery of naloxone, an opioid antagonist.

AIM: The feasibility of transdermal delivery of naloxone, an opioid antagonist, by anodal iontophoresis patches using Ag/AgCl electrodes was investigated. METHODS: To examine the effect of current strength, species variation and drug concentration on skin permeability of naloxone, in vitro skin permeation studies were performed using rat dorsal skin and porcine ear skin as the membrane. To determine in vivo transdermal absorption rate of naloxone, the iontophoretic patch system was applied to the dorsal skin of conscious rat with a constant current supply for 24h. RESULTS: The in vitro steady-state skin permeation flux of naloxone current-proportionally (0-360 µA/cm(2)) increased without significant differences between these two different skin types. The in vitro delivery rate through the porcine skin was found to be independent of the concentration of naloxone hydrochloride dehydrate in the donor patch over the range from 1 to 10% (w/v). In the in vivo pharmacokinetic study, plasma concentrations of naloxone steadily increased and sustained steady-state levels from 4h to 24h after the initiation of current application. In vivo steady-state transdermal absorption rates at 90 and 180 µA/cm(2) were 136 and 305 µg/h/cm(2), respectively. CONCLUSION: These results suggest that the transdermal delivery rates of naloxone by anodal iontophoresis are sufficient for the management of intoxication in opioid-overdosed patients.

Automated experimental system capturing three behavioral components during murine forced swim test.

AIMS: An automated experimental system applying a commercially available video image analyzer was developed for the simultaneous detection and measurement of three behavioral components; immobility, swimming (horizontal movements) and climbing (vertical movements) that occur in the murine forced swim test (FST). The system was validated using four typical antidepressants. MAIN METHODS: System validity was confirmed by demonstrating no significant difference in 6 min time course of control group and imipramine-dosed group (30 mg/kg) between manual examinations and automated digital analysis for all the three behaviors (i.e., correlation coefficients were 0.96, 0.83 and 0.94 for immobility, swimming and climbing, respectively). The effects of acute single treatment with four antidepressants in clinical use, i.e., imipramine, desipramine, bupropion and fluvoxamine were evaluated at doses of 15, 30 and 60 mg/kg using the system. KEY FINDINGS: In 2-4 min time span analysis, all four antidepressants reduced immobility and increased climbing significantly, desipramine and bupropion increased swimming significantly, while imipramine and fluvoxamine did not. SIGNIFICANCE: The automated experimental system enabled efficient and accurate analysis of the three murine behaviors during FST at once. Climbing could be more sensitive parameter to detect anti-depressant-like effect than immobility in this system.

An automated evaluation system for analyzing antinociceptive effects on intracolonic capsaicin-induced visceral pain-related licking behavior in mice.

INTRODUCTION: The development of automated detection systems for animal behaviors is increasing in value in terms of saving time, objective analysis and reducing the need for well-trained experimenters. SCLABA(®) (Noveltec Inc., Kobe, Japan) is a commercially available analysis system originally developed for analyzing scratching behaviors in rodents, based on distances between points in videotaped images. Here, we used this software to automate analysis of abdominal licking behavior associated with visceral pain in mice. METHODS: Yellow and green spots were applied to the snout and the lower abdominal region of mice respectively to provide reference points for automated analysis of video recordings. Abdominal licking behavior after intracolonic administration of 0.3% capsaicin solution as a measure of visceral pain was determined based on changes in the inter-spot distance. RESULTS: A distance threshold between the colored spots was chosen based on manual measurements showing that 99% of minimal distances were below this threshold. Using this threshold, the number of licks determined by the automated analysis significantly and positively correlated with that determined by manual observation (R(2)=0.95 and p<0.001). The neurokinin-1 receptor antagonist GR205171A dose-dependently inhibited capsaicin-induced licking detected by automated analysis. DISCUSSION: We demonstrated that visceral pain-related licking behaviors after intracolonic capsaicin treatment can be automatically detected by applying commercially available image analysis software. This automated experimental system is very efficient and useful to evaluate antinociceptive effect of a test compound on visceral pain.

In-vitro and in-vivo transdermal iontophoretic delivery of tramadol, a centrally acting analgesic.

OBJECTIVES: The feasibility of transdermal delivery of tramadol, a centrally acting analgesic, by anodal iontophoresis using Ag/AgCl electrodes was investigated in vitro and in vivo. METHODS: To examine the effect of species variation and current strength on skin permeability of tramadol, in-vitro skin permeation studies were performed using porcine ear skin, guinea-pig abdominal skin and hairless mouse abdominal skin as the membrane. In an in-vivo pharmacokinetic study, an iontophoretic patch system was applied to the abdominal skin of conscious guinea pigs with a constant current supply (250 µA/cm(2)) for 6 h. An intravenous injection group to determine the pharmacokinetic parameters for estimation of the transdermal absorption rate in guinea pigs was also included. KEY FINDINGS: The in-vitro steady-state skin permeation flux of tramadol current-dependently increased without significant differences among the three different skin types. In the in-vivo pharmacokinetic study, plasma concentrations of tramadol steadily increased and reached steady state (336 ng/ml) 3 h after initiation of current supply, and the in-vivo steady-state transdermal absorption rate was 499 µg/cm(2) per h as calculated by a constrained numeric deconvolution method. CONCLUSIONS: The present study reveals that anodal iontophoresis provides current-controlled transdermal delivery of tramadol without significant interspecies differences, and enables the delivery of therapeutic amounts of tramadol.

Inverse relationship between Sec14l3 mRNA/protein expression and allergic airway inflammation.

Bronchial asthma is an inflammatory disease of the airways. The Sec14l3 gene, encoding a 45-kDa secretory protein, is specifically expressed in airway epithelium. Here, we report on the kinetics of Sec14l3 expression following allergic inflammation of the lung. Brown Norway rats were sensitized by intraperitoneal injection of ovalbumin, followed by challenge with aerosolized ovalbumin after a 3-week interval. This animal model showed many features similar to human allergic asthma: an increase in inflammatory cells such as eosinophils, lymphocytes and neutrophils in bronchoalveolar lavage (BAL) fluid and histopathological alteration of lung tissue, exhibiting infiltration of these inflammatory cells and degeneration and necrosis of alveolar epithelium. These parameters reached their maximal level 24h after allergen challenge. In contrast, quantitative polymerase chain reaction analyses demonstrated a rapid and significant reduction of Sec14l3 mRNA in lung tissue and maximum reduction (to 1.4% of the control) was observed at 24h. Pretreatment with dexamethasone significantly suppressed both the Sec14I3 mRNA reduction and all of the inflammatory changes. The 45-kDa secretory protein was identified in the supernatant of BAL fluids. Two-dimensional gel images of the supernatant proteome also revealed down-regulation of the protein following inflammation (to approximately 30% of the control at 24h). Thus, Sec14l3 expression is highly and inversely associated with the progression of airway inflammation. Sec14l3 mRNA and protein may function in the homeostasis of airway epithelial cells under normal conditions.

Optimized analysis of the forced swim test using an automated experimental system: detailed time course study in mice.

INTRODUCTION: In most publications on the forced swim test studies, duration of immobility is measured during the last 4 min of the 6-min testing period as a marker for depression. However, it is not clear if 4-min span best captures antidepressant-like drug effects. In the present study, six typical antidepressants in clinical use were evaluated over time. Imipramine (60 mg/kg, PO), desipramine (60 mg/kg, PO), reboxetine (20 mg/kg, IP), bupropion (60 mg/kg, PO), fluoxetine (20 mg/kg, PO) and fluvoxamine (60 mg/kg, PO) were characterized over 1-min intervals for the 6-min testing period in order to identify more sensitive periods than the last 4-min span to detect drug effects, using the automated experimental system. METHODS: One day before the testing, wire rings were attached to the hind paws of male CD-1 mice. On the test day, after attaching a small magnet to the wire rings, each animal was placed for 6 min in a glass cylinder filled with water, which is surrounded by a coil. The duration of immobility was measured by the previously validated automated detection system, MicroAct, i.e. electrical signals generated by limb movements served as the marker for swimming behavior. RESULTS: In the 1-min interval examination, it was only in 2-3-min and 3-4-min spans that all of the six antidepressants reduced the duration of immobility with statistical significance. In the 2-4-min analysis, all of the six antidepressants demonstrated statistically significant reduction in the duration of immobility, while in the 2-6-min analysis, the reduction by fluvoxamine was not statistically significant. DISCUSSION: The detailed time course analysis of the FST in mice using the automated system revealed that the duration of immobility between 2 and 4 min is the optimum period to capture the antidepressant-like effect of test compounds.

Quantitative image analysis in adipose tissue using an automated image analysis system: differential effects of peroxisome proliferator-activated receptor-alpha and -gamma agonist on white and brown adipose tissue morphology in AKR obese and db/db diabetic mice.

Morphometric analysis of adipocytes is widely used to demonstrate the effects of antiobesity drugs or anti-diabetic drugs on adipose tissues. However, adipocyte morphometry has been quantitatively performed by manual object extraction using conventional image analysis systems. The authors have developed an automated quantitative image analysis method for adipose tissues using an innovative object-based quantitative image analysis system (eCognition). Using this system, it has been shown quantitatively that morphological features of adipose tissues of mice treated with peroxisome proliferator-activated receptor (PPAR) agonists differ dramatically depending on the type of PPAR agonist. Marked alteration of morphological characteristics of brown adipose tissue (BAT) treated with GI259578A, a PPAR-alpha agonist, was observed in AKR/J (AKR) obese mice. Furthermore, there was a 22.8% decrease in the mean size of adipocytes in white adipose tissue (WAT) compared with vehicle. In diabetic db/db mice, the PPAR-gamma agonist GW347845X decreased the mean size of adipocytes in WAT by 15.4% compared with vehicle. In contrast to changes in WAT, GW347845X increased the mean size of adipocytes in BAT greatly by 96.1% compared with vehicle. These findings suggest that GI259578A may activate fatty acid oxidation in BAT and that GW347845X may cause adipocyte differentiation in WAT and enhancement of lipid storage in BAT.