Animal Groups and Experimental Design
Twenty-four adult female Wistar rats weighing 200–240 g were purchased from Bio-LASCO Taiwan (Taipei, Taiwan). They were housed at a constant temperature under a consistent light-dark cycle (light from 07:00 to 18:00) at the Experimental Animal Center of National Taiwan Normal University. All animal surgical and experimental procedures were approved by the Institutional Animal Care and Use Committee of National Taiwan Normal University, College of Life Science (approval No. 106049) and performed in accordance with the guidelines of the National Science Council of the Republic of China (NSC 1997).
The rats were divided into four groups: (1) sham control (sham, n = 6), (2) 4 weeks of bladder ischemia induced by BPAO (BPAO, n = 6), (3) ADSCs injection through one of the femoral arteries in the week following BPAO and a total of 4 weeks of BPAO-induced bladder ischemia (BPAO + ADSCs, n = 6), and (4) ADSC-derived MVs injection through one of the femoral arteries at the week following BPAO and a total of 4 weeks of BPAO-induced bladder ischemia (BPAO + ADSC-derived MVs, n = 6). Baseline physiological parameters, including urinary frequency, water intake, food intake, urine output (labeled as urine), and feces output (labeled as feces), were recorded and analyzed in R-2100 metabolic cages (Lab Products, MD, USA) for 24 h in the four groups. Figure 1a illustrates the induction of the BPAO. The detailed procedures have been described in our previous study [16]. After BPAO, each rat received subcutaneous administration of ketoprofen (Sigma-Aldrich, Darmstadt, GER) 5 mg/kg once daily for one week. Four weeks after BPAO, the bladder microcirculation was evaluated. After physiological experiments with the metabolic cage, transcystogram, and pelvic nerve activity studies, the bladder was removed. The weight of the bladder was measured. One part of the bladder was cut and reserved for formalin fixation and further staining. Some of the samples were stored at − 70°C for protein expression analysis through western blotting and lipid peroxidation (malondialdehyde [MDA]) assay. The details of the respective techniques and studies are addressed in later sections.
Preparation of ADSCs and ADSC-Derived MVs
Adult female Wistar rats weighing 280–300 g were purchased from Bio-LASCO Taiwan (Taipei, Taiwan). The flank adipose tissue was collected under sterile conditions after administering general anesthesia. ADSCs standard isolation was performed as described previously in detail [17]. The adipose tissue was then stirred to aspirate the saline and oil phases. The fat was washed three to five times with 0.1 M phosphate-buffered saline (PBS) (Sigma-Aldrich, Darmstadt, Germany), and the lower phase was discarded until clear. Collagenase was added after the final upper phase was collected. The solution was incubated for 1–4 h at 37°C on a shaker. Next,10% fetal bovine serum (FBS) (Invitrogen, MA, USA) was added to the tube to neutralize collagenase. The fluid in the tube containing the digested fat was centrifuged at 800× g for 10 min. The supernatant containing liquid, lipid, and floating adipocytes was aspirated to obtain the left stromal vascular fraction (SVF) pellet. We used 160 mM NH4Cl to suspend the SVF pellet, which was incubated for 10 min at room temperature (RT). The pellet was centrifuged at 400× g for 10 min at RT after incubation. The final pelleted fraction of mononuclear cells was resuspended in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma-Aldrich, Darmstadt, Germany) supplemented with 40% FBS, penicillin–streptomycin, and 10 ng/mL of epidermal growth factor (Invitrogen, MA, USA) and incubated on a petri dish overnight to select adherent cells. The remaining debris and cells were removed the next day, and the plate was washed with PBS. The ADSCs were then maintained in low-glucose DMEM supplemented with 10% FBS, 1% penicillin–streptomycin, and L-glutamine at 5% CO2 and 37°C. ADSCs were maintained in a T75 flask and passaged until 80–90% confluence. ADSCs were trypsinized and counted to obtain 5 × 105 cells for treatment.
Preparation of ADSC-derived MVs
ADSC-derived MVs were isolated as previously described [17, 18]. Briefly, MVs were purified by differential ultracentrifugation. ADSCs (5 × 105 cells) were cultured in low-glucose DMEM without FBS and supplemented with 0.5% BSA overnight. The clear conditioned medium was transferred to centrifuge tubes and centrifuged for 10 min at 300× g and 4°C. The pellets filled with dead cells and cell debris were removed; the supernatant was retained. The supernatant was then centrifuged three times for 10 min at 2000× g, 30 min at 10,000× g, and 30 min at 10,000× g at 4°C. The final supernatant was discarded, and isolated MVs were suspended in Dulbecco’s phosphate-buffered saline (DPBS) (Sigma-Aldrich, Darmstadt, Germany) and then re-centrifuged for 30 min at 10,000× g at 4°C to remove contaminating proteins. We collected the pellet containing MVs and resuspended them in DPBS. MVs were washed and ultra-centrifuged at 10,000× g for 60 min at 4°C. The supernatant was discarded; the MVs pellets were resuspended in DPBS for later use. MVs purity was assayed with CD29 (Becton Dickinson, NJ, USA) and morphology was analyzed using a scanning electron microscope (JEOL-6500F, JEOL, Japan). The purified vesicles under this protocol yielded high CD29-positive expression, spheroid morphology, and 70 nm size exosomes by scanning electron microscopy [16]. ADSC-derived MVs were injected through the femoral artery at a dose of 100 µg MV proteins, and approximately 5 × 105 ADSCs were released overnight.
Measurement of Bladder Microcirculation
A full-field laser perfusion imager (Moor FLPI; Moor Instruments, Devon, UK) was used to quantify the intensity of microcirculatory blood flow in the bladder in the four groups 4 weeks after the experiments. The details were described previously [16]. This imager uses real-time laser speckle contrast continuous non-contact imaging, which exploits the random speckle pattern generated when the tissue is illuminated by laser light. The microcirculatory blood flow intensity in the region of interest was recorded as “Flux” with perfusion units. This is related to the concentration and average speed of moving red blood cells in the region of interest. When there is a high level of movement (fast flow), the changing pattern becomes more blurred, and the contrast in that region is reduced accordingly. Therefore, low contrast is related to high flow, and high contrast is related to low flow. The contrast image produces 16-color images that are correlated with blood flow in the field of interest. A low level of blood flow was recorded in blue, whereas a high level of blood flow was recorded in red color (range, 0–1000). Images were recorded and analyzed in real time using Moor FLPI 3.0 software (Moor Instruments, Devon, UK).
Evaluation of Transcystometrogram and pelvic nerve activity
All animals were anesthetized with subcutaneous urethane, which is known to allow full-reflex bladder contractions [19]. We used a transcystometrogram to evaluate variations in micturition parameters. Briefly, rats were anesthetized by subcutaneous injection of urethane (1.2 g/kg body weight). The bladder was exposed through a lower abdominal midline incision. A PE-50 catheter was then inserted through the apex of the bladder dome and connected through a T-tube to a P23 ID infusion pump and pressure transducer (Gould-Statham, MA, USA). During the experiment, the infusion rate was maintained at 1.2 mL/h, and intravesical pressure (IVP) was continuously recorded using an ADI system (Power-Lab/16S, ADI Instruments, Pty., Ltd., Castle Hill, Australia). The bladder was emptied. Three reproducible micturition cycles were recorded following bladder emptying. The following parameters of bladder activity were recorded: intercontraction interval (ICI), the time between two micturition cycles identified based on active contractions (> 15 mmHg), average urine amount per micturition (labeled as average urine amount), bladder volume, amplitude of IVP on micturition contraction (labeled as amplitude), and residual urine amount (labeled as residual urine). Additionally, we recorded and analyzed contraction phases during a micturition cycle: an initial rise in IVP (phase 1) and a series of high-frequency oscillations (HFOs; phase 2). Non-voiding contractions (NVCs) were characterized by an increase in pressure of > 20% from baseline, defined as the bladder pressure immediately before voiding. This was observed as an increase in pressure, which did not result in voids [20]. The number, mean amplitude, and maximum amplitude were analyzed.
The electrophysiological techniques used to record pelvic afferent nerve activity (PANA) and pelvic efferent nerve activity (PENA) have been described previously [19, 21]. Briefly, these activities were recorded simultaneously from two vesical branches of the pelvic nerve branch, which were micro-dissected from the surface of the bladder. The firing activities of each neural filament were recorded by placing the nerve fibers in parallel with two pairs of thin bipolar stainless steel electrodes. The electrodes and bladder nerves were bathed in a pool of warm (37°C) paraffin oil to prevent nerve drying. PANA was recorded after crushing the nerve central to the recording site to eliminate efferent firing. PENA was recorded when the bladder nerve was crushed distal to the recording site. The electrical signals were amplified 20,000-fold and filtered (high-frequency cut-off at 3000 Hz, low-frequency cut-off at 30 Hz) using a Grass model P511 a.c. preamplifier (Valley View, OH U.S.A.) and continuously recorded and displayed on an ADI system (Power-Lab/16S, ADI Instruments, Pty., Ltd., Castle Hill, Australia). The amplified signals (spikes) were transformed by a window discriminator 121 (World Precision Instruments, Sarasota, FL), which was set to count the total number of spikes per second. The background activity, which could be caused by nerve contact with electrodes, nerve damage during handling, and the equipment itself, was excluded from the window discriminator by adjustment of the threshold voltage. PE-50 catheters were placed in the left femoral artery to measure arterial blood pressure (ABP). ABP was recorded in an ADI system (Power-Lab/16S; ADI Instruments, Pty., Ltd., Castle Hill, Australia) with a transducer (Gould-Statham; Quincy, MA, USA). Three reproducible micturition cycles on the transcystogram were simultaneously recorded following bladder emptying. The following parameters were recorded: ABP (mmHg), IVP (mmHg), PENA(µv), PANA(µv), cumulated PENA (cPENA) (spikes/s), and cumulated PANA (cPANA) (spikes/s). The voltage changes (%) of PENA (µv) and PANA (µv), cPENA (spikes/s), and cPANA(spikes/s) between the micturition phase and baseline were analyzed.
Morphological Staining
A portion of the bladder was cut and fixed in a 10% neutral-buffered formalin solution. The tissues were dehydrated in graded ethanol and embedded in paraffin. Sections (4 µm) of the bladder were stained with hematoxylin and eosin to evaluate the extent of neutrophils. To prepare slides for immunohistochemical examination, slides were blocked with 5% bovine serum (Cat# ALB001; BioShop, Ontario, Canada) in PBS with 0.3% Triton-X 100 (Sigma-Aldrich, MO, USA) for 1 h. The slides were incubated overnight with primary antibodies prepared using 1% normal donkey serum and 0.1% Triton-X 100 in 0.1 M PBS at RT. Collagen I was labeled using a rabbit monoclonal antibody (bs-10423R; Bioss, MA, USA) purified at a working dilution of 1:250. The secondary antibody goat anti-rabbit IgG H&L (NEF812001EA; Invitrogen, MA, USA) was then applied at a dilution of 1:500. The samples were counter-stained with haematoxylin. Nerve growth factor (NGF) was labeled using a rabbit monoclonal antibody (ab52918; Abcam, Cambridge, UK) purified at a working dilution of 1:250. The secondary antibody goat anti-rabbit IgG H&L (NEF812001EA; Invitrogen, MA, USA) was then applied at a dilution of 1:500. The samples were counter-stained with haematoxylin. Antigen retrieval was performed using Tris-EDTA buffer (pH 9.0). PBS was used instead of primary antibody as a negative control.
Western Blot and Biochemical Evaluation
Expression levels of purinergic P2X2 and P2X3 receptors, muscarinic M2 and M3 receptors, NGF, and collagen-I were analyzed by western blotting as described previously [22–24]. Bladder samples were homogenized using a prechilled mortar and pestle in an extraction buffer containing 10 mM Tris-HCl (pH 7.6), 140 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 1% NP-40, 0.5% deoxycholate, 2% β-mercaptoethanol, 10 mg/mL pepstatin A, and 10 mg/mL aprotinin. The mixture was completely homogenized by vortexing and maintained at 4°C for 30 min. The homogenate was centrifuged at 12,000× g for 20 min at 4°C. The supernatant was collected; the protein concentration was determined using a Bio-Rad protein assay kit (BioRad Laboratories, CA, USA). Antibodies raised against P2X2 receptors (ab10266; Abcam, Cambridge, UK), P2X3 receptors (RA141399; Neuromics, MN, USA), M2 receptors (nb120-2805; Novus Biologi-cals, CO, USA), M3 receptors (ab87199; Abcam, Cambridge, UK), NGF (ab6199; Abcam, Cambridge, UK), and collagen-1 (bs-10423R; Bioss, MA, USA) were used. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis was performed using 10% separation gels in the absence of urea and Coomassie brilliant blue staining. For immunoblotting, proteins were transferred to Immobilon polyvinylidene difluoride membranes (Millipore, MA, USA) for 18 h at 100 mA in a Miniprotean III transfer tank (BioRad, CA, USA). Immunoreactive bands were detected by incubation with the aforementioned antibodies, followed by incubation with an alkaline phosphatase-labeled secondary antibody and western Lightning Plus-ECL stock solution (PerkinElmer, MA, USA) for 1 min at RT. The density of the band was semi-quantitatively determined by densitometry using an image analysis system (ImageJ; National Institutes of Health, USA). MDA concentration was determined using a lipid peroxidation assay kit (ab118970; BioVision, CA, USA).
Statistical Analysis
GraphPad Prism 6 (GraphPad Software, CA, USA) was used. All values are expressed as the mean ± standard error of the mean. Differences between groups were examined using one-way analysis of variance, followed by Duncan’s multiple-range test. Differences within the groups were examined using paired t-tests. Differences were considered significant at p < 0.05.