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

Histological Observations In control animals, nNOS positive neurons were widely distributed in the muscular laminae, with the majority of them located at the base of bladder and compara

CHAPTER III

RESULTS

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

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 2 weeks whence the bladder weight was 3.1 times that of

Fig 1 Photograph shows a big urinary bladder stones

(about 10mm in length) removed from an obstructed urinary bladder Bar=1mm

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 2 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 4 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)

(bladder/body weight)

Normal control 3 0.30 ± 0.027 0.00077 ± 0.00006 2-week PBOO 3 0.93 ± 0.165* 0.00243 ± 0.00045** 4-week PBOO 3 1.28 ± 0.070** 0.00297 ± 0.00031** 6-week PBOO 3 1.40 ± 0.279** 0.00300 ± 0.00066** 8-week PBOO 4 1.52 ± 0.275** 0.00308 ± 0.00050** 12-week PBOO 4 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<0.05, compared to normal control **p<0.01, compared to

normal control

Fig 2 Pictures show normal urinary bladder (A)

and 4-week obstructed urinary bladder (B) Note hypertrophic change after obstruction Arrows indicate urinary bladder.

RESULTS 87

3 Histological Observations

In control animals, nNOS positive neurons were widely distributed in the

muscular laminae, with the majority of them located at the base of bladder and

comparatively less so at body and even fewer at dome (Figs 3A, 3B & 3C) These

neurons often displayed long processes, some of which showed varicosities Nerve

processes tended to form bundles and networks linking different ganglia of varying

sizes (Fig 3D)

The majority of the nNOS cells were localized in ganglia of different sizes

containing 2-30 neurons each The large ganglia usually composed of 20-30 cells

each, located preferentially at the base of urinary bladder Smaller ganglia

containing fewer neurons were distributed mainly in the body and dome (Figs 3E &

3F)

NADPH-d staining showed a similar pattern of intramural ganglia (Figs 4 and

5) Numerous NADPH-d positive neurons were observed to be distributed in

muscular lamina Again, more ganglia were located at urinary bladder base than at

body and dome as illustrated in Fig 5C (dome) and 5D (base) In addition,

NADPH-d positive neurons showed a marked gradation of reactivity for the enzyme,

some of them being heavily stained while the remainder being moderately or weakly

stained (Fig 5) NADPH-d positive fibers tended to occur in bundles linking the

ganglia (Figs 4 and 5)

Beginning 2 weeks after urethral obstruction, some nNOS positive neurons

appeared to undergo degenerative changes: this consisted of irregular cell outline

(Figs 6A & 6B), vacuolated cytoplasm (Figs 6C & 6D), and eruption of cytoplasm

from the cell surface (Figs 6E & 6F) Occasionally, some cells showed signs of total

lysis (Figs 6G & 6H)

A feature worthy of note was that at 12 weeks while most cells exhibited

normal nNOS immunoreactivity (Fig 7A), some neurons were intensely stained for

nNOS (Fig 7B); such a feature with enhanced immunoreactivity was absent in the

controls

Fig 3 A, B and C showing intramural ganglia in the urinary bladder base (A), body (B) and

dome (C) of normal control guinea pig Note the larger concentration of ganglia at the base compared to the body and dome D is a higher magnification photomicrograph showing a few nNOS positive neurons at the bladder base The cells display long extending processes Some of the varicose fibers appear to form bundles and networks linking the neighboring ganglia E shows a large ganglion containing about 30 cells at the bladder base of normal guinea pig and

F showing much smaller ganglia of 3-4 neurons each at bladder body in normal animal Bar = 500µm (A, B and C); 125µm (D, E and F)

RESULTS 89

Fig 4 Lower magnification (A, C, E at 4x objective and B, D, F at 10x objective) showing an

overview of distribution of NADPH-d positive staining A and B show distribution of NADPH-d positive ganglia at the bladder base; C and D at the body; E and F at the dome Note the preferential distribution of NADPH-d positive neurons at the base Bar=500µm (A, C and E); 250µm (B, D and F)

Fig 5 NADPH-d positive ganglia in the urinary bladder of normal guinea pig A and B show

the same cluster of ganglia at different magnification (20x and 40x objective, respectively) at the base Note heavily stained (arrows) neurons compared to moderately (thick arrows) and weakly (arrowheads, in C and D) stained neurons A larger concentration of ganglia is observed at the base (A, B and D) compared to the body and dome (C shows a representative field of dome) In addition, nerve bundles are observed linking the ganglia Bar = 125µm (A); 50µm (B, C and D)

RESULTS 91

Fig 6 A and B showing intramural ganglion cells with irregular outlines (refer to Fig 3 normal

control)at the bladder base (A) and body (B) in animals killed at 8 and 6 weeks, respectively, after urethral obstruction C and D are intramural ganglion cells at the bladder base in animals killed at 2 (C) and 4 (D) weeks, respectively, after urethral obstruction Note the vacuolation of cytoplasm (arrows) E and F showing intramural ganglion cells at the bladder base (E) and body (F) in animals killed at 8 and 12 weeks, respectively, after urethral obstruction Note vacuolation and eruption of cytoplasm of cells (arrows) G and H are photomicrographs showing lysis of intramural ganglion cells (arrows) in the bladder base (G) and body (H) in animals killed at 8 weeks after urethral obstruction Bar = 50µm

Fig 7 Intensely stained neurons (arrows) are admixed with the moderately stained neurons

(arrowhead) at the urinary bladder base of 12 weeks after urethral obstruction Bar = 250µm (A); 50µm (B)

4 Cell Counts

Results of nNOS-IR intramural neurons in all 100µm thick sections derived

from the whole urinary bladder are shown in Table 2 Abercrombie formula was

used for correction of overestimation of nNOS positive intramural neurons in raw

count (Table 2) At 2 weeks after PBOO, the total number of nNOS positive neurons

decreased The reduction was most drastic at 4 weeks (Table 2) with a 60.6%

reduction in number compared with the control At 6 weeks, however, the number of

RESULTS 93

nNOS positive neurons restored gradually so that by 12 weeks post-obstruction the

number of nNOS positive neurons was almost reaching that of control (Fig 8)

Number of nNOS positive neurons Group n

Raw count Corrected number

Table 2 Raw count and corrected number (after Abercrombie’s correction) of nNOS positive

neurons (mean ± SD) in whole urinary bladder in control and obstructed groups Note: *p<0.05, compared to control **p<0.01, compared to control

5 Cell Size

The mean sectional area of nNOS positive neurons in the control was 463.18

(±169.62) µm2 Details are given in Table 3 The neuronal size was significantly

increased at 4 weeks and was further enhanced at 12 weeks (Fig 9)

Group n Average area of nNOS positive neurons (µm 2 )

Table 3 Average sectional area of nNOS positive neurons (mean ± SD) in control, 4- and

12-week PBOO groups Note: *p<0.01, compared to control

Fig 8 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

RESULTS 95

6 Electron Microscopic Observations

6.1 Conventional Electron Microscopic Observations

In semi-thin sections, all the intramural ganglia in normal guinea pigs were

located between muscle bundles A variable number of glial cells were associated

with the neuronal somata (Fig 10)

Ultra-structurally, the intramural neurons had a large, round, pale nucleus

bearing one or more conspicuous nucleoli (Fig 11) The neuronal cytoplasm

contained Golgi apparatus located in the peri-nuclear region (Fig 12) Associated

with the Golgi saccules were some small clear vesicles and membrane-bound

granular vesicles (Fig 12) The cisternae of rough endoplasmic reticulum (rER)

were present as short profiles (Fig 12) Mitochondria were randomly distributed

(Fig 12) The neuronal soma was surrounded by glial cells which had a smaller and

elongated nucleus 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)

Fig 9 B shows hypertrophic

nNOS positive neurons in 12-week PBOO bladder when compared to neurons in normal control bladder (A) Bar=50µm

Fig 10 Semi-thin sections of normal urinary bladder base stained with methylene blue Arrows

indicate intramural ganglion cells with a characteristic big, pale and round nucleus containing conspicuous nucleoli (dashed arrows) A glial cell (arrowhead) is closely associated with the surface of the ganglion cell Blood vessels (BV) and bundle of nerve fibers (thick arrow) are located around the ganglion cells M: muscle bundles Bar=50µm

RESULTS 97

Fig 11 Electron micrographs show intramural neurons in normal urinary base Panel A shows

a ganglion cell and its associated satellite cell The intramural neuron with its characteristically large, round, pale nucleus (labeled with “N”) is surrounded by a glial satellite cell bearing a smaller, darker nucleus (labeled with G) characterized by heterochromatin clumps Arrow indicates a portion of a satellite cell whose cytoplasm is lighter than that of neuron It appears

to enwrap the entire soma of the neuron Panel B shows portion of another intramural neuron at

a higher magnification Note the pale nucleus (N) and abundant 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 (arrowheads) are abundant and randomly distributed Dashed arrow indicates the cytoplasm of an associated glial cell Note the pale nucleus (N) Bar=600nm

Fig 13 Electron micrograph shows axonal profiles (arrows) near the soma of intramural

neurons whose nucleus is labeled with “N” “G” indicates the nucleus of a glial cell in close apposition to the neuronal soma “S” is the nucleus of a Schwann cell associated with an axon Bar=5µm

Fig 14 Electron micrograph shows two axons containing predominately clear vesicles (arrows)

in the axoplasm Arrowhead indicates a synapse Bar=0.5µm

RESULTS 99

6.2 Immunoelectron Microscopic Observations

nNOS immunoreaction product was deposited in various sub-cellular organelles, especially the mitochondria (Fig 15A) or distributed randomly in the

cytoplasm (Fig 15B) Some axonal profiles also contained DAB reaction product in

their plasma (Fig 15C)

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 membrane (arrows) “N”, nucleus

Panel B shows 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

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 Bar=2µm for A and B; =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 101

7 TUNEL

TUNEL labeling was carried out to detect possible apoptosis of the intramural

neurons in the urinary bladder after PBOO TUNEL labeled cells were occasionally

detected in both normal and 4-week PBOO urinary bladder There was no

significant difference in terms of frequency of labeled cells between the two groups

(Fig 18)

The identification of TUNEL labeled cells was confirmed by triple labeling, i.e

TUNEL+nNOS+DAPI which revealed that the apoptotic cells exhibited nNOS

immunofluorescence As with TUNEL labeling alone, the number of triple labeled

neurons in both the control and experimental groups was not significantly different

(Fig 19)

8 Real-time RT-PCR

The analysis of real-time RT-PCR showed that nNOS mRNA level decreased

significantly at 4 weeks after obstruction compared with that of the sham-operated

This is consistent with results by immunohistochemistry The mRNA level at 4-week

post-obstruction was 0.576 fold that of 4-week sham-operated animals reflecting a

decline of 42.4% (Fig 20)

Fig 18 TUNEL labeling in normal (A) and 4-week PBOO (B) urinary bladder In both normal

and obstructed animals, sporadic TUNEL labeled cells are observed Bar=25µm