Non adrenergic non cholinergic innervation of lower urinary tract after partial bladder outlet obstruction in guinea pig 2

EFFECTS OF L-ARGININE AND L-NAME TREATMENTS ON EXPRESSION OF nNOS IN THE GUINEA PIG URINARY BLADDER AFTER PBOO 1.. All animals subjected to PBOO showed bladder hypertrophy as reflected b

II EFFECTS OF L-ARGININE AND L-NAME TREATMENTS ON EXPRESSION OF nNOS IN THE GUINEA PIG URINARY BLADDER AFTER PBOO

1 Bladder Weight

The bladder weight represented the actual weight of the organ immediately upon its removal from the body and blotted dry (Table 4) All animals subjected to PBOO showed bladder hypertrophy as reflected by the significant increase in bladder weight, regardless of saline or drug treatment, when compared with the sham-operated control The value of sham-operated control was consistent with the previous part of present study Bladders were heaviest in the saline and L-arginine treated animals; however, no significant difference between these two groups was observed In the L-NAME treated animals, the increase in bladder weight was not as high when compared with the saline and L-arginine treated groups

To exclude the effects of body weight variance on the bladder weight, the value

of the bladder weight was corrected against the animal’s body weight and expressed

as ratio between the two (Table 4) The bladder/body weight ratio showed the same trend of increase as bladder wet weight, when compared with sham-operated animals The bladder/body weight ratio in the L-arginine treated animals was moderately decreased when compared with that of saline treated animals (0.00308 to 0.00324), but the difference was not statistically significant When compared between the saline and L-NAME treated groups, the bladder/body weight ratio in the

latter group (0.00252) was markedly reduced (p<0.05) by about 22%.

Group n Bladder weight

(g)

Ratio (bladder/body weight)

4-week-sham 5 0.326 ± 0.044 0.00079 ± 0.00011 4-week-PBOO+saline 5 1.190 ± 0.225* 0.00324 ± 0.00032* 4-week-PBOO+L-arginine 5 1.200 ± 0.168* 0.00308 ± 0.00034* 4-week-PBOO+L-NAME 5 0.746 ± 0.069* 0.00252 ± 0.00029*

Table 4 Bladder weights and ratio of bladder to body weight for sham-operated control and

saline or drug treatment groups (mean ± SD) *p<0.05, compared to 4w-sham-operated control p<0.05, compared to 4w-PBOO with saline treated control.

2 Cell Counts

The results of raw data on cell counting are shown in Fig 25A

In this connection, Abercrombie’s formula was used for correction of overestimation of numbers of nNOS positive intramural neurons (Fig 25B)

In sham-operated animals, the total number of nNOS positive neurons ranged from 2,865 to 3,162 per urinary bladder There was no significant difference between the 2-week-sham (3,162±367.6) and 4-week-sham (2,865±467) animals The result is comparable to the results of previous part of present study

Comparison between sham operated and saline treated PBOO animals showed that there was significant decrease in nNOS positive neurons in the latter group both

at 2- and 4-week

In animals with PBOO and given L-NAME injections for 2-week, the number

of nNOS positive neurons was significantly reduced from 2,011±347.1 in the control animals (2-week-saline) to 1,288±205.4 (2-week-L-NAME) However, in animals given L-arginine injection for 2-week, the number of nNOS positive neurons was significantly increased (2,948±404.6) Compared with the 4-week -saline injected

controls (1,199±245.2), 4-week-L-arginine treated animals maintained a significant increase in nNOS positive neurons (3,099±534.7) However, the neuronal number (1,323±275.7) remained relatively unchanged in the 4-week-L-NAME treated group

A

B

Fig 25 Panel A shows number of nNOS positive neurons (mean ± SD) in whole urinary bladder

in control and experimental groups Panel B shows number of nNOS positive neurons (mean ± SD) in whole urinary bladder in control and experimental groups after correction with Abercrombie’s formula.

*p< 0.05, compared to corresponding saline injected control.

**p<0.05, compared to corresponding sham-operated control.

3 Histological Observations

In control animals, nNOS positive neurons were observed in the muscular laminae, with some of them bearing long extending processes being intensely stained (Fig 26A); others showed a moderate staining (Fig 26B) Occasional nNOS positive neurons exhibited signs of degenerative changes as reflected by the vacuolated cytoplasm (Fig 26C) Some neurons were closely associated with the blood vessels that were weakly stained (Fig 26D), and in some areas, they appeared

to creep along the outer vascular wall (Fig 26E)

In L-arginine treated animals, there was a marked increase in nNOS expression

in intramural neurons both in cell numbers (Fig 27A) and immunoreactivity (Fig 27B) when compared with the saline treated (Fig 27D) or L-NAME treated animals (Fig 27E) Occasional neurons whose cytoplasm was vacuolated were observed (Fig 27C) Another striking feature in L-arginine treated animals was the occurrence

of many well-defined blood vessels ranging from about 10µm to 400µm in diameter (Fig 28A) when compared with the saline treated control (Fig 28B) On closer examination, the well-defined vascular outline was delineated by nNOS immunoreactive nerve fibers (Fig 28C) In larger calibred blood vessels, nNOS positive nerve fibers appeared to form meshes enwrapping the blood vessel (Fig 28D) The nerve fibers appeared to emanate from adjacent nNOS positive ganglion cells (Fig 28E)

In L-NAME treated animals, besides the significantly lower in number of nNOS positive neurons when compared with the L-arginine treated animals, the blood vessels as delineated by nNOS positive staining was less evident (Fig 28F)

Fig 26 Intramural ganglia in the urinary bladder of saline treated guinea pigs killed after

4-week PBOO: A Clusters of intensely stained intramural neurons for nNOS (arrows) with long extending processes are distributed between muscle fiber bundles (mf); B A small ganglion showing weak to moderate staining for nNOS; C Some neurons immunoreactive for nNOS show vacuolation of cytoplasm (arrows) Arrowheads indicate normal and intensely stained neurons

in the same ganglion D nNOS positive neurons and their associated blood vessels; E Some nNOS positive intramural ganglion cells and their tapering processes appear to perch on the blood vessels BV: blood vessel Bar =125µm.

Fig 27 A, D & E are representative views of nNOS immunoreactive ganglion cells in L-arginine,

saline and L-NAME treated animals’ urinary bladder, respectively Note the occurrence of large number of nNOS positive neurons in L-arginine treated animal, some of them are intensely stained Note also the paucity of nNOS positive ganglion cells and moderate staining in saline and L-NAME treated animals B is an enlarged view of ganglion cells in L-arginine treated animal C Arrows indicate nNOS positive cells whose cytoplasm appears vacuolated in L-arginine treated animal BV: blood vessel Bar =125µm (A, C-E) or =50 µm (B).

Fig 28 In L-arginine treated animals, vascular profiles are markedly accentuated (A) as

compared to that in saline treated animals (B) The vascular profiles are outlined by bundles of nNOS positive nerve fibers that enwrap the vascular walls (C) In larger calibred blood vessel, the nerve fiber bundles form prominent meshwork on the vascular surface (D) At a higher magnification, the bundles of nerve fibers appear to originate from associated ganglion cells (E).

In L-NAME treated animals (F), vascular outlines are hardly detected Arrowheads in F indicate moderately stained ganglion cells A, C-E: L-arginine treated; B: saline treated; F: L-NAME treated BV: blood vessel NFB: nerve fiber bundle Bar =125µm.

4 Western Blot Analysis

There is a significant increase (52%) in densitometric values for nNOS protein

in the L-arginine treated guinea pig bladder when compared with the saline treated group nNOS protein levels in L-NAME treated animals showed a moderate decrease (15%) when compared with the saline treated group, but the difference is not statistically significant (Fig 29)

Fig 29 Western blot analysis of nNOS expression in guinea pig bladder after 4 weeks PBOO

with saline or drug treatment The upper panel shows the typical image of guinea pig bladder sample immunoblotted with a monoclonal antibody against the human nNOS The guinea pig bladder sample showed 2 distinct bands approximately at 155 kDa and 135 kDa The upper band (155 kDa) is the expected size of nNOS protein and was used for OD measurement Lane 1: saline treated; lane 2: L-arginine treated and lane 3: L-NAME treated L-NAME treated animal shows a faint band compared with the saline treated and L-arginine treated animals The lower panel shows the results of Western blot analysis of nNOS protein expression Values are mean ±

SD * p< 0.05 vs saline treated control.

5 NO Colorimetric Assay

NO production in the bladder samples was significantly increased in 4-week-L-arginine treated animals and decreased in 4-week-L-NAME treated animals when compared with the saline treated group (Fig 30)

Fig 30 NO production in guinea pig bladder 4 weeks after PBOO with saline or drug treatment

as determined by NO colorimetric assay Data represent mean ± SD of the concentration of nitrate plus nitrite in bladder tissue which are stable reaction products of NO and have been used as a quantitative measure of NO production *Significant differences between control and drug treatment groups (p< 0.05)

6 Nitrotyrosine ELISA Test

Nitrotyrosine concentration increased slightly in the 4-week-L-arginine treated group, but the change was not statistically significant In 4-week-L-NAME treated group, nitrotyrosine decreased significantly by 46% when compared with the corresponding controls (Fig 31)

Fig 31 Nitrotyrosine amount in guinea pig bladder 4 weeks after PBOO with saline or drug

treatment as determined by nitrotyrosine ELISA test Data represent mean ± SD of the concentration of nitrotyrosine in bladder tissue *Significant differences between control and drug treatment group (p< 0.05)