Animal Groups and Experimental Design
Forty (40) 3-month old male Sprague-Dawley rats were obtained from Charles River Laboratories (Wilmington, MA, USA). All animal care, treatments and procedures were approved by the Institutional Animal Care and Use Committee of the University of California at San Francisco (AN080146-01).
Ten rats fed a diet of standard rat chow served as negative controls. The remaining 30 rats (obese hyperlipidemic rats, OHR) were fed a high-fat diet consisting of 2% cholesterol and 10% lard (Zeigler Brothers, Gardner, PA, USA). At five months, all rats in the negative control group and ten rats randomly selected from the OHR were placed in a metabolic cage for 24 hours, to perform voiding behavior studies.
After metabolic cage study, all rats underwent para-testicular fat harvest for procurement of ADSC. After culture and purification of ADSC the 30 OHR were divided into three groups: 1) injection of phosphate buffered saline (PBS) into the detrusor through a laparotomy (OHR+PBS), 2) Injection of ADSC via tail vein (OHR+TV), or 3) Injection of ADSC into the detrusor through a laparotomy (OHR+B). Negative control animals underwent laparotomy and PBS injection into the detrusor (NR+PBS).
One month after these treatments all rats underwent conscious cystometry. Animals were then euthanized and serum samples obtained for assessment of total cholesterol, high density lipoprotein (HDL), low density lipoprotein (LDL), triglyceride and glucose. Bladder tissues were harvested for immunohistochemical examination for Masson’s trichrome, 5-ethynyl-2′-deoxyuridine (EdU), α-smooth muscle actin (α-SMA), rat endothelial cell antigen-1 (RECA-1), and rat choline acetyltransferase (ChAT) staining.
All rats undergoing 24 hour voiding behavior studies were placed in a metabolic cage for 24 hours on a 12/12 hour dark/light photocycle.21
Each monitoring period started at 16:00. Micturition time and volumes were recorded via a fluid collector connected to a pressure transducer (Utah Medical Products, Midvale, UT, USA). The pressure transducer was connected to a Dell Pentium 4 computer with Laboratory View 6.0 software (National Instruments, Austin, TX, USA) to monitor the cumulative weight of the collected urine. All rats received water and food ad-libitum throughout the duration in the cage. Voiding frequency and volume were recorded for 24 hours.
Harvest and Processing of ADSC and EdU Labeling
Animals were anesthetized with isoflurane. A midline abdominal incision was made to expose the perigonadal fat pad. A specimen of para-testicular fat was harvested and placed in ice cold PBS. The animal was then closed in two layers with absorbable suture and anesthesia was weaned.
Fat tissue was rinsed with PBS in a 50 ml conical tube. Adipose tissue was removed from the upper layer to a fresh tube, and digested in 0.075% collagenase I (Sigma-Aldrich Cp., St Louis, MO, USA) for 1 hr at 37° C with shaking. The top lipid layer was removed and the remaining liquid portion was centrifuged at 220 × g for 10 min. The pellet was then treated with 160 mM NH4Cl for 10 min to lyse red blood cells. The remaining cells were suspended in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), and plated at a density of 1 × 106 cells in a 10-cm dish. The culture dish was placed in a 5% CO2 incubator for 3–5 days to allow the formation of ADSC colonies. For the purpose of cell tracking, ADSC were treated with 10 μM EdU (Invitrogen, Carlsbad, CA, USA) overnight. A total of 3 × 106 EdU-labeled ADSC were collected into a 2-ml conical tube containing 1 ml PBS; these ADSC were subsequently utilized for injection.
All animals were anesthetized with isoflurane and underwent midline laparotomy to expose the bladder. Animals underwent injection of 1 ml PBS (NR+PBS and OHR+PBS group), or 3 million of autologous ADSC in 1 mL PBS (OHR+B) into the bladder. Animals in the OHR+TV group received 3 million of autologous ADSC transplantation via the tail vein. After treatment the laparotomy was closed in two layers.
Placement of Bladder Catheters and Conscious Cystometry
Bladder catheters were implanted under isoflurane anesthesia 24 hours prior to conscious cystometry. The abdomen was opened through a midline incision and a polyethylene catheter (PE-90, Clay-Adams, Becton Dickinson, Parsippany, NJ, USA) was implanted into the bladder through the dome. The catheter was tunneled subcutaneously and brought out through a skin incision on the rat’s back.
Conscious cystometry was performed by placing an awake rat in a custom-made tunnel within a metabolic cage (Braintree Scientific, Braintree, MA, USA). Conscious cystometry enables the recording of micturition time and volumes via a fluid collector connected to a pressure transducer connected to surgically implanted bladder catheters (Utah Medical Products, Midvale, UT, USA). The bladder catheter was connected via a T-tube to both a pressure transducer (Utah Medical Products, Midvale, UT, USA) and an infusion pump (Harvard Model 22, KD Scientific, Holliston, MA, USA). After calibration of the pressure transducer to zero, the bladder was filled with room temperature normal saline at a rate of 0.1 mL/min, while recording simultaneous pressure on a sensor input module (model SCXI 1121, National Instruments, Austin, TX, USA) connected to a Dell Pentium 4 computer with Laboratory View 6.0 software (National Instruments). All rats were given around 10 min for the voiding patterns to stabilize. Thereafter micturition were recorded for 30 min .
After euthanasia, tissue samples were fixed in cold 2% formaldehyde and 0.002% picric acid in 0.1 M phosphate buffer, PH 8.0, for 4 hours followed by overnight immersion in buffer containing 30% sucrose. The specimens were then embedded in OCT Compound (American Master Tech Scientific, Inc, Lodi, CA, USA) and stored at 70°C until use. Sections were cut at 5 μm, mounted into charged slides and air dried for 5 min. The slides were stained with Masson’s trichrome for connective tissue and smooth muscle histology.
EdU staining with immunostaining for α-SMA was performed by placing slides in 0.3% H2O2/methanol for 10 min. Slides were then twice-washed in PBS for 5 min and incubated with 3% horse serum in PBS/0.3% Triton X-100 for 30 min at room temperature. After draining this solution from the tissue section, the slides were incubated at room temperature with anti-α-SMA antibody (Abcam Inc., Cambridge, MA, USA) overnight. After rinsing with PBS, sections were incubated with FITC-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA, USA). After three rinses with PBS, the slides were incubated with freshly made Click-iTTM reaction cocktail (Invitrogen, Carlsbad, CA, USA) for 30 min at room temperature without light followed by Dilute Hoechst (Invitrogen, Carlsbad, CA, USA) for nuclear staining.
We used RECA-1 as a marker for endothelial cells to study the changes in vascular profile and ChAT in cholinergic nerves profile. The tissue sections were similarly prepared as described above. After draining excess fluid, the sections was incubated overnight at room temperature with the following antibodies: mouse anti-RECA-1 (Abcam Inc, Cambridge, MA, USA) and goat anti-ChAT (Chemicon, Temecula, CA, USA). Immunostaining of the tissue was performed with the avidin-biotin-peroxidase method (Elite ABC, Vector Labs, Burlingame, CA, USA), with 3,3-diaminobenzidine as chromagen, followed by hematoxylin counterstain.
Image and Statistical Analysis
For image analysis, five randomly selected fields per slide were photographed and recorded at 200x magnification for Masson’s trichrome staining and at 400x magnification for RECA-1 and ChAT staining using a digital still camera (Nikon DXM1200) and ACT-1 software (Nikon Instruments Inc., Melville, NY, USA). Image-Pro Plus imaging software was used for quantification of differential staining (Media Cybernetics, Silver Spring, MD, USA). The percentage of smooth muscle was calculated as the sum of the smooth muscle (red stained) areas divided by the sum of all smooth muscle and connective tissue (blue stained) areas.15
Data was analyzed with Prism 4 (GraphPad Software, Inc., San Diego, CA, USA) and expressed as mean ± standard error of the mean for continuous variables. The continuous data was compared the groups using Student’s t-test and one-way analysis of variance. The Bonferroni test was used for post-hoc comparisons. Statistical significance was set at p < 0.05.