Combined estrogen and ghrelin administration decreases expression of p27(kip1) and proportion of isomyosin type I in the striated urethral and anal sphincters and levator ani of old ovariectomized rats.

We compared estrogen and/or ghrelin effects on pelvic floor muscles in old versus young adult ovariectomized rats. Ovariectomized Fisher 344 rats (18 and 3 months old, n = 24 x 2) received 42 daily intraperitoneal 17-beta estradiol (10 microg kg(-1)), ghrelin (2 microg kg(-1)), both, or vehicle (n = 6 x 4/group). Cytoplasmic p27(kip1) expression and isomyosin I proportion in striated urethral and anal sphincters and levator ani were measured, respectively, by Western blot analysis and gel electrophoresis with immunohistochemistry of muscle ghrelin receptors and radioimmunoassay of circulating growth hormone. In young adult rats, estrogen significantly decreased cytoplasmic p27(kip1) and isomyosin I signal intensities. In old rats, ghrelin and estrogen/ghrelin significantly decreased both intensities with greater estrogen/ghrelin effect. Ghrelin receptors were not immunostained in any muscle. Estrogen and/or ghrelin significantly increased or decreased, respectively, circulating growth hormone in old and young adult rats. Estrogen/ghrelin administration reversed pelvic floor muscle ageing changes in old ovariectomized rats through growth hormone production.

Combined estrogen and ghrelin administration restores number of blood vessels and collagen type I/III ratio in the urethral and anal canal submucosa of old ovariectomized rats.

We compare the effects of estrogen and/or ghrelin on vascular counts and collagen I/III ratio of urethral and anal canal submucosa in old vs young-adult ovariectomized rats. Ovariectomized Fisher 344 rats (18 and 3 months old, n = 24 x 2) received 42 daily intraperitoneal 17-ss estradiol (10 microg/kg), ghrelin (2 microg/kg), both, or vehicle (n = 6 x 4 per group). Blood vessel counts and collagen I/III ratio were measured, respectively, by light microscopy and Western blot analysis with immunohistochemistry of ghrelin receptors. Estrogen significantly increased urethral and anal vascular counts and collagen I/III ratio in young-adult rats. In old rats, only combined estrogen/ghrelin administration significantly increased both variables. This was not observed with estrogen or ghrelin separately. Ghrelin receptors were immunostained in urethral and anal submucosa of all samples. Combined estrogen/ghrelin administration restored postovariectomy urethral and anal canal submucosal vessel number and collagen I/III ratio in old rats suggesting independent ageing effect.

Enhancement of dissolution amount and in vivo bioavailability of itraconazole by complexation with beta-cyclodextrin using supercritical carbon dioxide.

The main objective of this study was to improve the inclusion formation between itraconazole and beta-cyclodextrin and thus enhance dissolution amount and bioavailability characteristics of itraconazole. Inclusion complexes between itraconazole and beta-cyclodextrin were prepared using simple physical mixing, conventional coprecipitation method, and supercritical carbon dioxide (SC CO(2)). Effects of process variables (temperature, pressure) and drug:cyclodextrin ratio on inclusion yield and thermal behavior of the solid complexes prepared by SC CO(2) were studied and compared to those obtained by physical mixing and coprecipitation methods. In addition, dissolution amounts of the products obtained by different methods were measured in gastric fluid. Finally, pharmacokinetic studies of the inclusion complexes were conducted in male Wistar rats to assess the bioavailability of the prepared complexes. Results showed that temperature, pressure and itraconazole:beta-cyclodextrin ratio had significant effects on the inclusion yield of the complex prepared by SC CO(2) method. Higher inclusion yields were obtained in the SC CO(2) method as compared to physical mixing and coprecipitation methods. In vivo drug pharmacokinetic studies showed that the itraconazole-beta-cyclodextrin product prepared using SC CO(2) gave higher bioavailability of itraconazole (in blood, liver and kidney of male Wistar rats) as compared to the products obtained by physical mixing or coprecipitation methods.

The effect of ovariectomy on biomarkers of urogenital ageing in old versus young adult rats.

The aim of this study was to compare the effects of ageing and ovariectomy on biomarkers of urogenital ageing in old and young-adult rats. Fisher 344 rats (18- and 3-months-old, n = 6 x 2) underwent ovariectomy. Age-matched sham animals received no intervention (n = 6 x 2). One month later, biomarkers of urogenital ageing were evaluated (light microscopic count of urethral and anal canal submucosal blood vessels, Western blot analysis of urethral, and anal canal submucosal collagen I and III and cytoplasmic p27(kip1) expression in the striated urethral and anal sphincters and levator ani and gel electrophoresis of isomyosin I proportion in these muscles) and compared in all groups (n = 24). All biomarkers of urogenital ageing studied were significantly increased in old compared to young-adult sham rats. Ovariectomy significantly increased these changes further in old versus young-adult rats with either smaller or larger differential effect than ageing compared to young-adult sham animals. Ovariectomy significantly exacerbates normative urogenital ageing changes in rats.

Estrogen and ghrelin decrease cytoplasmic expression of p27kip1, a cellular marker of ageing, in the striated anal sphincter and levator muscle of ovariectomized rats.

A study was carried out to investigate the effect of estrogen and/or ghrelin on the cellular marker of ageing, p27kip1, in pelvic floor muscles of ovariectomized rats. Virgin Wistar rats (13 months old) underwent ovariectomy followed (1 month) by 42 daily intraperitoneal 17-beta estradiol (10 microg/kg), ghrelin (2 microg/kg), both hormones, or placebo vehicle (n=6x4 groups). Six more age-matched animals underwent sham surgery without ovariectomy. Cytoplasmic expression of p27kip1 in the striated urethral and anal sphincters and levator muscle was measured by Western blot analysis in all animals (n=30). p27kip1 signal intensity significantly increased postovariectomy in all muscles compared to sham animals. In the anal sphincter and levator, signal intensity decreased to sham levels with ghrelin or estrogen and decreased further after estrogen or ghrelin and estrogen/ghrelin administration. Urethral sphincter signal intensity decreased without reaching sham levels after drug administration. Estrogen and/or ghrelin replacement reverses the ovariectomy-induced exacerbation of biochemical cellular ageing in the anal sphincter and levator muscle of middle-aged rats.

Estrogen and ghrelin increase number of submucosal urethral and anal canal blood vessels in ovariectomized rats.

OBJECTIVES: Urinary and fecal control deteriorates after menopause, but it is not clear whether this is age or hormone related. This study investigates whether administration of estrogen and/or the anti-aging growth hormone-releasing peptide, ghrelin, improves the adverse effects of menopause/aging on urethral and anal canal submucosal blood vessel counts in middle-age rats. METHODS: Female Wistar rats (13 months old) underwent ovariectomy, followed 1 month later by intraperitoneal once-daily administration of 17-beta estradiol (10 microg/kg), ghrelin (2 microg/kg), both hormones, or vehicle (n = 6 in each of four groups) for 42 days. An age-matched sham group (n = 6) received no intervention. Submucosal blood vessels were counted by light microscopy in five randomly selected fields from five nonconsecutive sections (5 microm thick) per rat of formalin-fixed and paraffin-embedded tissue blocks of the urethra and anal canal stained with hematoxylin-eosin. The results are expressed as the mean vessel number per high power field (x400). RESULTS: Ovariectomy significantly reduced submucosal urethral and anal vascular counts below the sham values (7.41 +/- 0.98 versus 5.46 +/- 0.82, P = 0.003 and 7.16 +/- 1.11 versus 4.92 +/- 0.65, P = 0.0009, respectively). Estrogen restored the urethral counts (7.76 +/- 0.88, P = 0.5) and ghrelin or combined estrogen and ghrelin administration significantly increased the counts to greater than the sham counts (8.68 +/- 0.99, P = 0.04 and 9.72 +/- 1.21, P = 0.004, respectively). Estrogen, ghrelin, and combined estrogen and ghrelin administration also restored the anal counts to sham levels (7.26 +/- 0.97, P = 0.8; 6.56 +/- 0.78, P = 0.3; and 7.76 +/- 0.88, P = 0.3, respectively). CONCLUSIONS: Combined or individual replacement of estrogen and ghrelin produces a beneficial effect by reversing the ovariectomy-induced decrease in urethral and anal canal submucosal vessel numbers in middle-age rats.