Non adrenergic non cholinergic innervation of lower urinary tract after partial bladder outlet obstruction in guinea pig 1

84 RESULTS CHAPTER III RESULTS 85 RESULTS I. EFFECTS OF PBOO ON nNOS EXPRESSION IN THE INTRAMURAL GANGLIA OF THE GUINEA PIG URINARY BLADDER 1. Behavior Observations and Stone Formation in the Obstructed Urinary Bladder Under random observation, guinea pigs underwent PBOO were observed to have frequent and difficult micturition associated with decreased flow rate and even strangury. Haematuria is a common symptom occurred in the PBOO animals. When sacrificed, all animals with haematuria showed vesical calculi which ranged from sand-like particles to stones of 0.5mm to 10mm diameter (Fig. 1). The incidence of bladder stone is about 46% (40 out of 87 obstructed animals). All these symptoms are consistent with that of clinical observation in patients with BPH. The sham-operated and normal animals did not show any change in micturiton pattern or haematuria and bladder stone. Fig. 1. Photograph shows a big urinary bladder stones (about 10mm in length) removed from an obstructed urinary bladder. Bar=1mm. 2. Bladder Weight The bladder weight represented the actual weight of the organ immediately upon its removal from the body and blotted dry. The value of the bladder weight was also corrected against the animal’s body weight and expressed as ratio between the two (Table 1). Following urethral obstruction, the bladder weight showed a steady increase which was first noted at weeks whence the bladder weight was 3.1 times that of 86 RESULTS the normal control, and by 12 weeks, the increase was 6.4 folds (Table 1; Fig. 2). To exclude the possibility that the increase in bladder weight following urethral obstruction was not due to increase in body weight, the organ weight was also expressed as the ratio of bladder to body weight. In all the experimental animals with urethral obstruction, the ratio was significantly increased, indicating that the increase in bladder weight was attributed to bladder hypertrophy, rather than normal growth of bladder. At weeks post-obstruction, the ratio increased from 0.00077 (normal ratio) to 0.00243 reflecting a significant increase of 3.16 times. The increase continued to be drastic till weeks where it was 3.86 times that of normal value. This ratio increase sustained right up till the 12th week, abated at a slower pace beyond the 4th week (3.9, 4.0 and 4.1 times at 6th, 8th and 12th weeks, respectively) (Table 1). Fig. 2. Pictures show normal urinary bladder (A) and 4-week obstructed urinary bladder (B). Note hypertrophic change after obstruction. Arrows indicate urinary bladder. Group n Bladder weight (g) Normal control 0.30 ± 0.027 Ratio (bladder/body weight) 0.00077 ± 0.00006 2-week PBOO 0.93 ± 0.165* 0.00243 ± 0.00045** 4-week PBOO 1.28 ± 0.070** 0.00297 ± 0.00031** 6-week PBOO 1.40 ± 0.279** 0.00300 ± 0.00066** 8-week PBOO 1.52 ± 0.275** 0.00308 ± 0.00050** 12-week PBOO 1.92 ± 0.269** 0.00313 ± 0.00049** Table 1. Bladder weights and ratio of bladder to body weight for normal control and obstructed groups (mean ± SD). Note: *p[...]... showing dense chromatin masses (Fig 11 ) Nerve fibers consisting of a varying number of axons were found in the connective tissue of urinary bladder Some nerve fibers were in close proximity to the intramural ganglion cells (Fig 13 ) The axons contained predominantly clear vesicles (Fig 14 ) 96 RESULTS Fig 10 Semi-thin sections of normal urinary bladder base stained with methylene blue Arrows indicate intramural... Differences in number of nNOS positive intramural ganglion cells at the base of urinary bladder of normal (A), 4-week PBOO (B) and 12 -week PBOO (C) guinea pigs Note the significant decrease in nNOS positive neurons in 4-week PBOO animal compared with normal and 12 -week PBOO animals Bar=250µm 95 RESULTS Fig 9 B shows hypertrophic nNOS positive neurons in 12 -week PBOO bladder when compared to neurons in normal... =0.5µm for C and D Fig 17 Electron micrograph shows dilated rER (arrows) in a degenerating intramural ganglion cell in 4-week obstructed guinea pig Note the immunoreaction product is deposited on the membranes of rER and mitochondria (arrowhead) Bar=0.5µm RESULTS 10 1 7 TUNEL TUNEL labeling was carried out to detect possible apoptosis of the intramural neurons in the urinary bladder after PBOO TUNEL labeled... organelles in the cytoplasm The thick arrow indicates the nucleolus Bar=6µm (A); 2µm (B) Fig 12 Electron micrograph shows portion of a typical intramural neuron in normal urinary bladder of guinea pig The neuronal cytoplasm contains Golgi apparatus located in the peri-nuclear region (thick arrow) Associated with the Golgi saccules are some small clear vesicles Arrows indicate cisternae of rER Mitochondria... was determined on the basis of a quantitative densitometric estimation of the 15 5 kDa band immuno-detected by Western blot and compared among control (normal and 4-week sham-operated, n=5), 4-week (n=5) and 12 -week (n=5) PBOO animals Fig 24 (top panel) shows the typical images of guinea pig urinary bladder sample immuno-blotted with a monoclonal antibody against the human nNOS The guinea pig bladder. .. showed two distinct bands at approximately 15 5 kDa and 13 5 kDa The upper band (15 5 kDa) is the expected size of nNOS protein and used for OD measurement The bottom panel shows the results of Western blot analysis of nNOS protein expression The levels of 15 5 kDa nNOS protein were significantly RESULTS 10 5 lower in the 4-week obstructed group compared to the control (4-week obstructed: 0.375±0 .11 2 OD; control:... portion of another nNOS-IR intramural neuron in which DAB immunoreaction product is localized in the cytosol (arrows) Panel C shows an axon containing nNOS immunoreaction product RESULTS 10 0 Fig 16 Electron micrographs show normal intramural neuron (A and C) and degenerating intramural neuron (B and D) in 4-week obstructed animals Note the highly swollen organelles in the cytoplasm of degenerating neuron... their plasma (Fig 15 C) At 4-week after PBOO, some neurons appeared to undergo degenerative changes including dilation of organelles and vacuolation of cytoplasm (Figs 16 and 17 ) Fig 15 Electron micrographs show nNOS immunoreaction product in the intramural neurons and axons Bar=0.5µm Panel A shows portion of an nNOS-IR intramural neuron in which DAB reaction product is localized in the mitochondrial... homogenates Upper panel is the representative blot Lane I: 12 -week obstructed; lane II: 4-week obstructed and lane III: control Two distinct bands at about 15 5kDa and 13 5kDa are observed The 15 5kDa band was used for OD measurement There is a significant decrease in densitometric values for nNOS protein in the urinary bladder of 4-week PBOO guinea pigs when compared to controls Values are mean ± SD *p . 85 I. EFFECTS OF PBOO ON nNOS EXPRESSION IN THE INTRAMURAL GANGLIA OF THE GUINEA PIG URINARY BLADDER 1. Behavior Observations and Stone Formation in the Obstructed Urinary Bladder Under. 532 .14 2-week PBOO 3 2,366 ± 260.97* 2,056 ± 229.65* 4-week PBOO 3 1, 342 ± 14 3 .13 ** 1, 1 81 ± 12 5.95** 6-week PBOO 3 1, 559 ± 45.74** 1, 372 ± 40.25** 8-week PBOO 4 1, 705 ± 17 0. 71* * 1, 500 ± 15 0.22**. masses (Fig. 11 ). Nerve fibers consisting of a varying number of axons were found in the connective tissue of urinary bladder. Some nerve fibers were in close proximity to the intramural ganglion