Kyu-Youn Ahn

Preparation of poly(dl-lactide-co-glycolide) microspheres encapsulating all-trans retinoic acid

Poly(DL-lactide-co-glycolide) (PLGA) microspheres containing all-trans retinoic acid (atRA) were prepared by o/w solvent evaporation method and various preparation parameters, such as poly(vinyl alcohol) (PVA) concentration in aqueous solution, PVA MW, drug weight, solvent, polymer MW, and polymer weight, on the characteristics of microspheres and drug release were investigated. PVA concentration in water phase was a critical factor in making microspheres consistently with smooth surface and round shape. In our study, at least 2% (w/v) of PVA in aqueous solution was necessary for making microspheres with round shape. The particle size of microspheres ranged 10-100 microm. AtRA was slowly released from PLGA microspheres over 30 days. Sterilization of microspheres by ethylene oxide (EO) gas at 37 degrees C did not significantly affect the characteristics of drug release or its morphology. Cell growth inhibition of atRA was affected by preparation process of microspheres rather than the EO-gas sterilization process. These results indicate that PLGA microspheres containing atRA are acceptable for controlled release devices for use in the treatment of brain tumor.

Brain tumor invasion model system using organotypic brain-slice culture as an alternative to in vivo model

Abstract Purpose. The primary cause of local recurrence and therapeutic failure in the treatment of malignant gliomas is the invasion of tumor cells into the surrounding normal brain. While it is known that malignant gliomas infiltrate diffusely into regions of normal brain, it is frequently very difficult to unequivocally identify the solitary invading glioma cell in histopathological preparations, or in experimental glioma models. We have developed an experimental invasion assay system, which allows us to track the solitary invasive glioma cell, using human brain tissue obtained from routine craniotomies for seizures or trauma. Methods. This tissue is cut into 1-mm thick slices and cultured in the upper chamber of Transwell culture dishes on top of a 0.4-µm pore size polyester membrane, which is fed on medium provided in the lower chamber. Glioma cells are stably transfected with vectors containing a green fluorescent protein (GFP) cDNA. Stable, high-level expression GFP transfectants were selected by direct visualization under fluorescence microscope. In addition, various tumor spheroids are stained with vital dye, DiI, to track the invading cells. GFP-expressing glioma cells or stained spheroids were then implanted on the center of the brain slice, and the degree of brain tumor invasion into the brain tissue was evaluated at different time points by optical sectioning using a confocal microscope. Results. We observed that GFP-expressing glioma cells or stained spheroids could be readily tracked and followed with this model system. Individual tumor cells that exhibited green or red fluorescence could be identified and their migration path through the brain slices unequivocally followed. Conclusion. This experimental invasion system may be of considerable utility in studying the process of brain tumor invasion and in evaluating its invasiveness in individual brain tumor because it not only provides a better representation of extracellular matrix molecules normally encountered by invading glioma cells, but also provides the fluorescent tag applied to the tumor cells.

Psx homeobox gene is X-linked and specifically expressed in trophoblast cells of mouse placenta.

We previously isolated a cDNA clone for a homeobox‐containing gene with its expression restricted to the extraembryonic tissues. In this study, Psx gene expression was further examined using in situhybridization to determine the cellular distribution of Psx transcripts during embryo development. Psx expression was first detected at embryonic day 8.5 only in trophoblast giant cells and chorionic ectoderm. At E 9.5 and E 13.5, the expression was restricted to the giant cells and the labyrinthine trophoblast layer. In addition, the gene expression was detected in differentiated Rcho‐1 trophoblast cells in vitro, which is typical of trophoblast giant cells in vivo, but not in proliferating Rcho‐1 cells and HRP‐1 cells. Interestingly, rat Psx homologue mRNA is about 200 bp shorter than mouse Psx, suggesting that there is a high degree of sequence divergence between the mouse and rat Psx homologues. The sequence divergence, perhaps as a result of rapid evolution, is further supported by the zoo blot analysis because the Psx gene was detectable only in mouse and rat but not in other vertebrate species tested. Psx is localized to the murine X chromosome. Taken together, our results suggest that Psx gene plays a unique role in the function of differentiated trophoblast cells and also serves as a useful model for studying trophoblast cell lineages and the rapid evolution of homeobox genes. Dev Dyn 1999;216:257–266. ©1999 Wiley‐Liss, Inc.

Tear production and ocular surface changes in experimental dry eye after elimination of desiccating stress.

PURPOSE To investigate the severity and duration of desiccating stress-induced dry eye disease between mice with and without a genetic predisposition to spontaneous autoimmunity. METHODS Experimental dry eye was induced in 12- to 16-week-old wild-type C57BL/6 and autoimmune NOD.B10.H2(b) mice by subcutaneous injection of scopolamine with exposure to an air draft for 10 days. Tear volume and corneal smoothness were measured at baseline, 5 and 10 days after desiccating stress, and 3, 7, 14, and 28 days after the removal of desiccating stress. Periodic acid-Schiff staining and immunohistochemistry were performed to evaluate the densities of conjunctival goblet cells and CD4(+) T cells in each group. Interleukin (IL)-1β and IL-6 concentrations in conjunctival tissues were measured by multiplex immunobead assay. RESULTS Signs of experimental dry eye were noted at 5 and 10 days after desiccating stress in both strains. After the removal of desiccating stress, in C57BL/6 mice, tear production and corneal smoothness improved at 3 and 7 days, respectively, and conjunctival goblet cells and CD4(+) T-cell densities and cytokine levels returned to baseline levels at 14 days. In contrast, in NOD.B10.H2(b) mice, none of the parameters recovered to baseline levels during a period of 28 days after the removal of desiccating stress. CONCLUSIONS After the removal of desiccating stress in experimental dry eye, tear volume and ocular surface parameters recovered within 2 weeks in C57BL/6 mice, whereas they remained unchanged in NOD mice. In contrast to autoimmune mice, experimental dry eye can be reversed after the elimination of desiccating stress in nonsusceptible mice.

Diabetes Mellitus Induces Vaginal Tissue Fibrosis by TGF-beta; 1 Expression in the Rat Model

The commonly reported sexual problem in women with diabetes mellitus is lack of vaginal lubrication. It is our hypothesis that reduced vaginal lubrication in diabetic women may result from the structural changes of the vagina. The aim of this study was to investigate in the diabetic rat model the vaginal structures using histochemistry and the expression of TGF- g 1 using immunohistochemistry. Twenty female Sprague-Dawley rats (200-210 g) were divided into two groups: control and experimental. The experimental group ( n = 10) received intravenous injection of streptozotocin (50 mg/kg). After 4 weeks, blood glucose levels were measured, and the vagina of the rat was excised. Serial sections of the vagina were used to perform hematoxylin and eosin (H & E) and Massons trichrome stains, and for immunohistochemistry to identify TGF- g 1 expression. The mean blood glucose concentrations were 67 - 11 mg/dL (range; 50-85) in the control group and 522 - 61 mg/dl (range; 429-590) in the experimental group. In the diabetic animals, vaginal tissue revealed reduced epithelial layers and decreased vaginal submucosal vasculatures compared to the control animals. The collagen connective tissue in the submucosal area of the diabetic animal tissue showed a dense and irregular, distorted arrangement. The immunoreactivity of TGF- g 1 in the diabetic animals was prominent in the collagen connective tissue, fibroblasts, and smooth muscle fibers, whereas no immunoactivity was detected in the vaginal structures of the control animals. Diabetes mellitus may induce vaginal tissue fibrosis by TGF- g 1 expression in the rat model. This implies that reduced vaginal lubrication in the diabetic women may result from the structural changes of the vagina.

Diabetes Induced Alteration of Clitoral Hemodynamics and Structure in The Rabbit

PURPOSE We investigated the effect of diabetes on clitoral hemodynamics and structures in the rabbit. MATERIALS AND METHODS A total of 25 New Zealand White female rabbits weighing 3 to 3.5 kg. were divided into 2 groups, including 5 in the control and 20 in the experimental group. Experimental animals received intravenous injection of alloxan hydrochloride (Sigma Chemical Co., St. Louis, Missouri) (100 mg./kg.). The development of diabetes was verified by measuring body weight and blood glucose levels. After 12 weeks clitoral cavernous blood flow in ml. per minute per 100 gm. tissue was measured with a laser Doppler flowmeter. Cross sections of the clitoris were used for histochemistry and histomorphometric image analysis. RESULTS After 12 weeks 5 animals were included in the diabetes group. Mean baseline flaccid and peak clitoral cavernous blood flow plus or minus standard deviation significantly decreased in the diabetic group compared with the control group (3.9 +/- 1.6 and 5.8 +/- 2.2 versus 7.2 +/- 2.5 and 12.9 +/- 5.8 ml. per minute per 100 gm. tissue, respectively, p <0.05). Histology revealed diffuse clitoral fibrosis in the diabetic group. On histomorphometry the mean proportion of clitoral cavernous smooth muscle in the diabetic group was significantly decreased compared with the control group (51.9% +/- 4.9% versus 62.3% +/- 3.1%, p < 0.05). CONCLUSIONS These results show that diabetes mellitus produces significant adverse effects on the hemodynamic mechanism of clitoral engorgement and leads to diffuse clitoral cavernous fibrosis. It implies that decreased sexual arousal in diabetic women may result from structural changes in the clitoris.

Effect of Estrogen Deprivation on the Expression of Aquaporins and Nitric Oxide Synthases in Rat Vagina

INTRODUCTION The expression of aquaporin (AQP) water channels in rat vagina was recently reported. AIM The purposes of this study were to investigate the effect of 17beta-estradiol on the expression of the AQP-1 and AQP-2 water channels and nitric oxide synthase (NOS) isoforms in rat vagina. METHODS Female Sprague-Dawley rats (230-240 g, N = 90) were divided into three groups: control (N = 30), bilateral ovariectomy (N = 30), and bilateral ovariectomy, followed by subcutaneous injections of 17beta-estradiol (50 microg/kg/day, N = 30). After 4 weeks, genital hemodynamics and vaginal secretions were measured after pelvic nerve stimulation, and the animals were then killed. The expression and cellular localization of AQP-1, AQP-2, endothelial NOS (e-NOS), and neuronal NOS (n-NOS) were determined in each group by immunohistochemistry and Western blot. MAIN OUTCOME MEASURES The expression and cellular localization of AQPs and NOS isoforms after estrogen deprivation. RESULTS Estimated vaginal secretions (mg, mean +/- standard error) were significantly lower in the ovariectomized group (2.9 +/- 0.62) than in the control group (5.7 +/- 1.25) and returned to the control value in the group after treatment with 17beta-estradiol (6.5 +/- 1.22) (P < 0.05). Both AQP-1 and e-NOS immunoreactivities were localized in the capillaries and venules of the lamina propria of the vagina, and n-NOS was expressed in the nerve fibers of the subepithelial lamina propria. The expression of AQP-2 was localized solely in the superficial layer of the vaginal epithelium. The protein expressions of AQP-2, e-NOS, and n-NOS were significantly lower after ovariectomy and were restored to the control level after 17beta-estradiol treatment. However, there was no significant change in AQP-1 expression. CONCLUSIONS Decreased vaginal secretion after estrogen deprivation may be partly due to functional changes in both AQPs and NOS isoforms in the vagina. The potential role of AQPs in water transport in the vagina might differ according to the type of AQP.

Expression of Aquaporin Water Channels in the Vagina in Premenopausal Women

INTRODUCTION Aquaporins (AQPs) are membrane proteins that facilitate water movement across biological membranes. This study builds on a previous report on the distinct localization of AQPs in the rat vagina. AIM The purposes of this study were to investigate the localization and expression of the AQPs in the vaginal tissue of premenopausal women. METHODS Anterior vaginal tissue was collected during transvaginal uterine myomectomy or hysterectomy from 10 premenopausal women (mean age, 40 years) for Western blot and immunohistochemistry. MAIN OUTCOME MEASURES The expression and cellular localization of AQP1-9 were determined in the human vagina by Western blot and immunohistochemistry. RESULTS Immunolabeling showed that AQP1 was mainly expressed in the capillaries and venules of the vagina, AQP2 was expressed in the cytoplasm of the epithelium, AQP3 was mainly associated with the plasma membrane of the vaginal epithelium, and both AQP5 and AQP6 were expressed in the cytoplasm throughout all vaginal epithelium. Western blot analysis revealed bands at 28 kDa for AQP1, 2, 3, 5, and 6 proteins. However, AQP4, 7, 8, and 9 were not detected. CONCLUSIONS The distinct localization of AQPs in the human vagina suggests that AQP1, 2, 3, 5, and 6 may play an important role in vaginal lubrication in women.

Identification and Characterization of a New Member of the Placental Prolactin-Like Protein-C (PLP-C) Subfamily, PLP-Cβ1

We have isolated a complementary DNA (cDNA) clone that encodes a new member of the PRL-like protein-C (PLP-C) subfamily of the PRL gene family. The clone was amplified from a 13.5-day-old mouse conceptus cDNA library by PCR using primers based on conserved regions of PLP-C sequences. The full-length cDNA encodes a predicted protein of 241 residues, which contains a putative signal sequence and 2 putative N-linked glycosylation sites. The predicted protein shares 55-66% amino acid identity with mouse PLP-Calpha and rat PLP-D, PLP-H, PLP-Cv, and PLP-C and also contains 6 homologously positioned cysteine residues. Thus, we named this protein PLP-Cbeta for consistency. We have also isolated rat PLP-Cbeta from rat placenta cDNA library. Surprisingly, two messenger RNA (mRNA) isoforms of rat PLP-Cbeta were isolated: one mRNA (rPLP-Cbeta) encodes a 241-amino acid product, but another mRNA (rPLP-Cbetadelta39) lacks 39 bases that encode for a region rich in aromatic amino acids. The 39-bp region corresponds to exon 3 of other PLP-C subfamily members, such as PLP-Calpha, PLP-Cv, and d/tPRP. It suggests that the two isoforms are probably generated by an alternative splicing from a single gene. RT-PCR analysis revealed that the rPLP-Cbeta form was dominantly expressed in placenta, although both isoforms are coexpressed during placentation. The mouse PLP-Cbeta mRNA expression, which was specific to the placenta, was first detected by Northern analysis on embryonic day 11.5 (E 11.5) and persisted until birth. However, in situ hybridization analysis revealed mPLP-Cbeta expression on E 10.5 in specific trophoblast subsets, such as giant cells and spongiotrophoblast cells. mPLP-Cbeta mRNA was detected in the labyrinthine zone on E 18.5, suggesting that spongiotrophoblast cells had penetrated the labyrinthotrophoblast zone. Consistent with the observed expression in trophoblast giant cells, PLP-Cbeta expression was also detected in in vitro differentiated Rcho-1 cells, which express the trophoblast giant cell phenotype. In summary, overall high amino acid identity (79%), the locations of cysteine residues, and consensus sites for N-linked glycosylation between mouse and rat PLP-Cbeta clearly indicate that PLP-Cbeta is a bona fide member of the PLP-C subfamily. The conservation between mouse and rat, the presence of alternative isoforms, and the pattern of expression during gestation suggest the biological significance of PLP-Cbeta during pregnancy.

COMP-angiopoietin-1 promotes cavernous angiogenesis in a type 2 diabetic rat model.

Cartilage oligomeric matrix protein-angiopoietin-1 (COMP-Ang1) is an angiogenic factor for vascular angiogenesis. The aim was to investigate the effect of an intracavernosal injection of COMP-Ang1 on cavernosal angiogenesis in a diabetic rat model. Male Otsuka Long-Evans Tokushima Fatty (OLETF) rats made up the experimental group (1 yr old) and Long-Evans Tokushima Otsuka (LETO) rats made up the control group. The experimental group was divided into vehicle only, 10 µg COMP-Ang1, and 20 µg COMP-Ang1. COMP-Ang1 was injected into the corpus cavernosum of the penis. After 4 weeks, the penile tissues of the rats were obtained for immunohistochemistry and Western blot analysis. The immunoreactivity of PECAM-1 and VEGF was increased in the COMP-Ang1 group compared with the vehicle only group. Moreover, the expression of PECAM-1 and VEGF was notably augmented in the 20 µg Comp Ang-1 group. In the immunoblotting study, the expression of PECAM-1 and VEGF protein was significantly less in the OLEFT rats than in the control LETO rats. However, this expression was restored to control level after intracavernosal injection of COMP-Ang1. These results show that an intracavernosal injection of COMP-Ang1 enhances cavernous angiogenesis by structurally reinforcing the cavernosal endothelium.