16 research outputs found
Functional and molecular characterization of hyposensitive underactive bladder tissue and urine in streptozotocin-induced diabetic rat
Background: The functional and molecular alterations of nerve growth factor (NGF) and Prostaglandin E2 (PGE2) and its receptors were studied in bladder and urine in streptozotocin (STZ)-induced diabetic rats. Methodology/Principal Findings: Diabetes mellitus was induced with a single dose of 45 mg/kg STZ Intraperitoneally (i.p) in female Sprague-Dawley rats. Continuous cystometrogram were performed on control rats and STZ treated rats at week 4 or 12 under urethane anesthesia. Bladder was then harvested for histology, expression of EP receptors and NGF by western blotting, PGE2 levels by ELISA, and detection of apoptosis by TUNEL staining. In addition, 4-hr urine was collected from all groups for urine levels of PGE2, and NGF assay. DM induced progressive increase of bladder weight, urine production, intercontraction interval (ICI) and residual urine in a time dependent fashion. Upregulation of Prostaglandin E receptor (EP)1 and EP3 receptors and downregulation of NGF expression, increase in urine NGF and decrease levels of urine PGE2 at week 12 was observed. The decrease in ICI by intravesical instillation of PGE2 was by 51% in control rats and 31.4% in DM group at week 12. Conclusions/Significance: DM induced hyposensitive underactive bladder which is characterized by increased inflammatory reaction, apoptosis, urine NGF levels, upregulation of EP1 and EP3 receptors and decreased bladder NGF and urine PGE2. The data suggest that EP3 receptor are potential targets in the treatment of diabetes induced underactive bladder. © 2014 Nirmal et al
TEM micrographs showing endocytosis mediated uptake of liposome encapsulated gold marker at 37°C (Panel A&C).
<p>Higher magnification image of inscribed area from panel A showed that vesicle like structures in endosomal compartment (marked by E) contained cluster of electron dense dark particles inside a single cell (Panel C). In contrast, only extracellular binding of liposomes containing dark grains was observed at 4°C (Panel B) and corresponding higher magnification image (Panel D) showed that vesicle like structures in a single cell were devoid of dark gold particles, which indicates absence of internalization due to temperature dependent inhibition of endocytosis. Gold encapsulated in liposomes is marked by red colored G inside a red circle, nucleus is marked by N, endocytic vesicles are marked by E and finger like projections called as lateral interdigitation are marked by LI. Inscribed area of panel A and B in white rectangle is magnified further in panel C and D, respectively and magnification is shown by scale bar in each panel.</p
Illustration depict temperature dependent endocytoic uptake of liposome encapsulated gold (Lipo-gold).
<p>The cellular process of clathrin and caveolin mediated endocytosis are energy dependent and therefore only occur at 37°C and are inhibited at 4°C. Compared to mhCD, chlorpomazine was more efficient in blocking endocytosis of liposomes, which indicates a predominance of clathrin mediated endocytosis as a mechanism of endocytosis.</p
TEM micrographs showing endocytosis mediated uptake of liposome encapsulated gold in rat bladder (A) as revealed by electron dense dark grains consistent with uptake of gold across the urothelium.
<p>(B) Dense black grains binding to the cell surface were noted in rat group instilled with colloidal gold in absence of liposomes. Gold encapsulated in liposomes are marked by red colored G inside a red circle and plain gold is marked by yellow G in the images. (C) Untreated rat bladder is marked by (absence of dark black gold grains. Magnification of 8900x was used in all the images and is shown by the scale bar.</p
Effect of different endocytic inhibitors on cellular uptake of fluorescent liposomes.
<p>Flow cytometry analysis suggest clathrin mediated endocytosis as the dominant pathway for the internalization of fluorescently labeled liposomes by UROtsa cells at 37°C. Chlorpromazine (inhibitor of clathrin) dose dependently inhibited the internalization as cell fluorescence from externally bound non-internalized liposomes was quenched by sodium dithionite. Fluorescence intensity of liposomes in cells untreated with inhibitors was taken as 100%.</p
Bladder histology, western blotting for detection of EP1, EP3, NGF, and ELISA for detection of PGE2.
<p>*P<0.05, and</p><p>**P<0.01 vs Control.</p
DM induced change of body weight, bladder weight, serum glucose level, and urine production.
<p>*P<0.05 vs control;</p><p>**P<0.0001 vs control;</p>@<p>P<0.05 vs 4 wk;</p>@@<p>P<0.0001 vs 4 wk.</p
Western blot analysis of EP1, EP3 receptors and NGF expression in the bladder (N = 6, per group).
<p>At DM week 12, the protein amount of EP3 was significantly increased (1.4 fold) and NGF was significantly decreased (60.6%) than the control rat. There was no significant change of EP1 receptors.</p
Photomicrographs of bladder sections (N = 6, per group).
<p>A, B, C: Control Group; D, E, F: DM week 4; G, H, I: DM week 12, (×200). A, D, G (H and E stain); B, E, H (trichome stain); CFI (TUNEL stain). DM rat at week 12 (G, H) showed increase of inflammatory reaction compared to the control rat (A, B). DM rat at week 4 (F) showed significant increase of TUNEL stain (9.4 fold) compared to the control rat (C).</p