Non adrenergic non cholinergic innervation of lower urinary tract after partial bladder outlet obstruction in guinea pig 4

Distribution CGRP immunoreactivity was localized in nerve fibers distributed throughout the urinary bladder.. Distribution of nerve fibers immunoreactive for CGRP and SP was comparable,

RESULTS 131

IV EFFECTS OF PBOO ON EXPRESSION OF SOME NEUROPEPTIDES IN URINARY BLADDER, DRG AND SPINAL CORD OF GUINEA PIG

1 CGRP and SP

1.1 Urinary Bladder

1.1.1 Distribution

CGRP immunoreactivity was localized in nerve fibers distributed throughout the urinary bladder Nerve trunk and varicose fibers showing CGRP immunoreactivity were found in the muscle layer (Fig 52A) In addition, some CGRP-IR nerve bundles run along the blood vessels or forming mesh-like work wrapping around them (Fig 53) A few intramural ganglia also showed CGRP immunoreactivity (Fig 54)

Like CGRP, SP immunoreactivity was detected in the nerve fibers (Fig 52B), associated with blood vessels and intramural ganglion cells (Fig 55) Distribution of nerve fibers immunoreactive for CGRP and SP was comparable, but the density of SP-IR nerve fibers as well as those associated with blood vessels and intramural ganglia was less when compared to that of CGRP-IR nerve fibers (Fig 52)

Fig 52 Distribution of CGRP- (A) and SP-immunoreactivity (B) in nerve fibers in

sham-operated guinea pig urinary bladder More CGRP-IR nerve fibers (+++) are found in the urinary bladder than SP-IR fibers (++) Bar=200µm.

Fig 53 CGRP-IR nerve bundles extend along blood vessel (A, arrows) or form network

wrapping the blood vessel (B, thick arrows) Arrowheads in A and B show thick CGRP-IR nerve trunk Bar=50µm (A) and 200µm (B).

Fig 54 CGRP-IR intramural ganglia in the guinea pig urinary bladder Arrows in A and B

indicate CGRP positive intramural ganglia/neurons Note the nerve bundles appear to emanate from the ganglia (A, arrowheads) At a higher magnification (B), the nerve fibers are distinguishable from the background by the bead-like appearance (thick arrows).

Fig 55 SP immunoreactivity in intramural ganglia (A, arrow) and perivascular nerve fiber

network (B, arrowhead) Note nerve bundles appear to arise from the intramural ganglia (thick arrows) in A Bar=100µm (A) and 200µm (B).

RESULTS 133

1.1.2 Changes in CGRP and SP Expression in the Bladder after PBOO

At 4 weeks after urethral obstruction, immuno-expression of CGRP (Fig 56)

as well as SP (Fig 57) was noticeably reduced in the nerve fibers of the bladder

Fig 56 CGRP-IR nerve fibers in urinary bladder of guinea pig A: 4-week sham (+++); B:

4-week PBOO (+) Note the dramatic decrease in immunoreactive nerve fibers at 4 weeks Bar=250 m.

Fig 57 SP-IR nerve fibers in urinary bladder of guinea pig A: 4-week sham (++); B: 4-week

PBOO (+) Note the dramatic decrease in immunoreactive nerve fibers at 4 weeks Bar=250 m.

1.2 DRG

1.2.1 Distribution

CGRP immunoreactivity was localized in both nerve fibers and cell bodies of neurons in DRG It is mainly found in small- and medium-sized neurons with moderate to intense staining The percentage of CGRP positive neurons was 37%±1.9% against the total DRG neurons In general, the large ganglion cells were unreactive for CGRP CGRP-IR nerve fibers were seen running in all directions, some in close proximity to the ganglion cells and others forming pericellular networks (Fig 58)

Some satellite cells were also CGRP positive, especially those surrounding the large-sized CGRP negative neurons (Fig 59) Immunoelectron microscopy confirmed the localization of CGRP immunoreactivity in satellite cells (Fig 60)

As with CGRP immunostaining, SP immunoreactivity was localized in randomly distributed small- to medium-sized ganglion cells and nerve fibers in DRG

SP positive neurons made up 27%±3.5% of the total DRG neurons The immunoreactivity varied from moderate to heavy staining Like the CGRP stained DRG, some nerve fibers originated from SP positive neurons were closely associated with the SP negative neuron Some satellite cells surrounding the neurons also appeared to be stained for SP (Fig 61)

Immunoelectron microscopy showed that the SP immunoreaction products were distributed throughout the ganglion cells associated with mitochondria and cisternae of rough endoplasmic reticulum (rER) (Fig 62) The reaction products were also found in nerve fibers between the ganglion cells as well as in some satellite cell (Fig 63)

RESULTS 135

Fig 58 CGRP immunoreactivity in L6 DRG of sham-operated guinea pig Arrows indicate

small-sized CGRP positive neurons Thick arrows indicate medium-sized CGRP-IR neurons Arrowheads indicate CGRP negative neurons; most of them are large-sized DRG neurons CGRP immunoreactivity is also distributed in nerve fibers coursing between the neurons This is evident at a higher magnification (C, dashed arrow) In C, a large CGRP negative neuron appears to be enveloped by a tortuous CGRP-IR nerve fiber (blue arrow) that originates from an adjacent CGRP positive neuron Arrowhead in C shows a CGRP negative neuron with CGRP positive pericellular nerve fiber network around it Bar=250µm (A), 125µm (B) and 50µm (C).

Fig 59 CGRP immunoreactivity is detected in some satellite cells (arrows) Bar=50µm.

Fig 60 Electron micrograph shows a CGRP-IR satellite cell (arrows) in close apposition to a

CGRP negative neuron in DRG NN, neuronal nucleus; SN, satellite cell nucleus Bar=2µm.

RESULTS 137

Fig 61 SP immunoreactivity in L6 DRG of sham-operated guinea pig Arrows indicate

small-or medium-sized SP positive neurons randomly distributed throughout the ganglion Arrowhead

in C indicates SP positive nerve fiber (thick arrows) making contact with a SP negative large neuron The SP positive fibers originate from a neighboring SP positive neuron The dashed arrow in B shows another SP negative neuron surrounded by SP positive satellite cells Bar=250µm (A), 125µm (B) and 50µm (C).

Fig 62 Electron micrograph shows distribution of SP immunoreaction products that are mainly

deposited on the subcellular organelles such as mitochondria and rER (arrows) N, nucleus of DRG neuron Bar=0.5µm.

Fig 63 Electron micrographs show SP-IR satellite cell (arrows) and nerve fibers (thick arrow).

NN, neuronal nucleus; SN, satellite cell nucleus Bar=5µm (A) and 1µm (B).

1.2.2 Changes in CGRP and SP Expression in the DRG after PBOO

The percentage of CGRP- and SP-IR neurons against total neurons in the L6-S1 DRG was markedly increased at 4 weeks after PBOO (Figs 64 and 65) The increase for CGRP positive neurons was from 36.6% to 43.1%; while that of SP was from 27.2% to 35.9% (Fig 66)

RESULTS 139

Fig 64 CGRP-IR neurons in 4-week sham (A) and 4-week PBOO (B) DRG (L6) Note more

CGRP positive neurons are detected in 4-week PBOO DRG when compared to 4-week sham operated animals Bar=250µm.

Fig 65 SP-IR neurons in 4-week sham (A) and 4-week PBOO (B) DRG (L6) Note more SP

positive neurons are detected in 4-week PBOO DRG when compared to 4-week sham operated animals Bar=250µm.

Fig 66 Bar chart shows percentage of CGRP- and SP-IR neurons against total neurons in L6

and S1 DRG (pooled) * (for CGRP) / (for SP) indicate p<0.05 when compared with corresponding sham-operated animals.

1.3 Spinal Cord

1.3.1 Distribution

CGRP immunoreactivity in the spinal cord was characterized by the dense terminal plexus in the superficial laminae of the DH and nerve bundles running along the lateral edge of the DH to the region of the lateral horn Smaller nerve fibers with punctate immunostaining were observed to scatter throughout the deeper laminae (Fig 67) Abundant CGRP-IR fibers formed bundles which in some cases connected the deeper part the DH towards the central canal (Fig 67) No CGRP-IR cell bodies were found in the DH

In the VH, large cells believed to be motoneurons as determined by their external morphology and location were stained for CGRP (Fig 68)

SP-IR nerve fibers were present mainly in superficial laminae of the DH, with a few fibers in deeper laminae The nerve bundles in deeper part of the DH were not

as apparent as those seen in CGRP immunostaining The main difference in distribution of SP immunoreactivity, when compared to that of CGRP, was that there were numerous SP-IR nerve fibers concentrated in area dorsal to the central canal (Fig 69) Some neurons in the VH were found to be stained moderately for SP (Fig 69B)

RESULTS 141

Fig 67 CGRP immunostaining in the DH (S1-2) of normal guinea pig at S1-2 level CGRP

immunoreactivity is localized in nerve fibers and varicosities concentrated in the superficial laminae and nerve bundles (arrows) running along the lateral edge of the DH to the region of the lateral horn Smaller immunopositive nerve fibers are also scattered throughout deeper laminae (dashed arrows) Abundant CGRP-IR nerve fibers form bundles which in some cases connect the deeper part the DH towards the central canal (thick arrows) CC: central canal Bar=125µm (A) and 50µm (B and C).

Fig 68 CGRP-IR neurons (arrows) in VH of spinal cord of normal guinea pig CC, central

canal Bar=250µm (A) and 50µm (B).

Fig 69 SP immunoreactivity in the lumbosacral spinal cord (L6-S1) of normal guinea pig The

SP-IR nerve fibers are found in superficial laminae of DH with a few fibers in deeper laminae Note the dot-like immunostained nerve fibers (thick arrows) concentrated in area dorsal to the central canal (CC) Some VH neurons are also immunoreactive for SP (arrows, B) Bar=250µm.

1.3.2 Changes in CGRP and SP Expression in the Spinal Cord after PBOO

At 4 weeks after PBOO, immuno-expression for CGRP and SP in DH was markedly enhanced when compared to the corresponding 4-week sham-operated animals (Figs 70 and 71) Measurement of density of immunoreactivity in DH of spinal cord confirmed this histological observation (Fig 72)

Fig 70 CGRP immunostaining in the lumbosacral spinal cord (L6-S1) A: 4-week

sham-operated; B: 4-week PBOO Note the increased CGRP immunoreactivity in DH at 4-week PBOO Bar=250µm.

RESULTS 143

Fig 71 SP immunostaining in the lumbosacral spinal cord (S1-2) A: 4-week sham-operated; B:

4-week PBOO Note the increased immunoreactivity in DH at 4-week PBOO Bar=500µm.

Fig 72 Figure shows quantitative comparison of CGRP and SP immunoreactivity in DH of L6

and S1 spinal cord before and after 4-week PBOO * (for CGRP) / (for SP) indicate p<0.05 when compared with corresponding sham-operated animals.

2 VIP

2.1 Urinary Bladder

2.1.1 Distribution

VIP immunoreactivity was localized in nerve fibers that accompanied the muscle bundles Single VIP-IR nerve fiber and coarse nerve trunk could be observed throughout the bladder muscular laminae (Fig 73) Occasionally, VIP-IR intramural ganglia were found (Fig 73)

A striking feature in the distribution of VIP immunoreactivity was that some blood vessels were wrapped by fine VIP positive nerve fibers forming a mesh work

on the surface of the vascular wall (Fig 74)

Fig 73 VIP immunoreactivity in the urinary bladder of normal guinea pig VIP-IR nerve fibers

(arrows) as well as coarse nerve trunk (thick arrows) could be found in muscle layer Occasionally, VIP positive intramural ganglia can be seen (arrowhead) Bar=100µm (A) and 50µm (B).

Fig 74 VIP-IR nerve fibers wrapping around the blood vessel as a neural network At a higher

magnification (B), some of the stained nerve fibers appear to be beaded Bar=200µm (A) and 50µm (B).

2.1.2 Changes in VIP Expression in the Urinary Bladder after PBOO

After 4-week PBOO, the VIP immunoreactivity in the urinary bladder was markedly reduced when compared to 4-week sham-operated animals (Fig 75)

RESULTS 145

Fig 75 VIP immunoreactivity in the urinary bladder of guinea pig A shows VIP-IR fibers

(arrows) in 4-week sham (+++) and B shows VIP-IR fibers in 4-week PBOO (+) Note the dramatic decrease in VIP-IR nerve fibers in 4-week PBOO urinary bladder C shows that after 4-week PBOO, the intense nerve mesh work around the blood vessel (BV) becomes less intense (arrow in C) when compared to that in normal bladder (refer to Fig 74) Thick arrow in C indicates a VIP positive ganglion closely associated with blood vessels Bar=100µm.

2.2 DRG

VIP positive neurons were observed mainly in the small- to medium-sized neurons in the DRG Some mildly stained VIP DRG neurons appeared to be encircled by satellite cells that were intensely stained for VIP (Fig 76) No apparent change was observed in the VIP immunoreactivity in the DRG following PBOO

2.3 Spinal Cord

2.3.1 Distribution

VIP immunoreactivity was mainly observed in nerve fibers located in the superficial laminae (Fig 77A) At the sacral level, VIP immunreactivity was also found in the nerve bundle running along the lateral edge of the DH to the region of

Fig 76 VIP immunoreactivity in S1 DRG of

normal guinea pig Arrows indicate VIP positive neurons which are small to medium-sized Some weakly stained large DRG neurons appear to be encircled by VIP positive satellite cells (thick arrows) Bar=50µm.

the lateral horn (Fig 78) In the VH, there were only a few neurons lightly stained with VIP (Fig 77B)

Fig 77 VIP immunoreactivity in the lumbosacral spinal cord (L6) of normal guinea pig A

shows that VIP immunoreactivity in nerve fibers (arrows) is mainly distributed in the superficial laminae B shows several lightly stained neurons in the VH CC, central canal Bar=250µm.

2.3.2 Changes in VIP Expression in the Spinal Cord after PBOO

At 4 weeks after PBOO, VIP immuno-expression enhanced significantly when compared with 4-week sham-operated animals (Figs 78 and 79)

Fig 78 VIP immunostaining in the lumbosacral spinal cord (S1-2) A: 4-week sham-operated;

B: 4-week PBOO Note the increased immunoreactivity in DH of 4-week PBOO CC, central canal Arrows indicate the nerve bundle running along the lateral edge of the DH to the region

of the lateral horn (LH) Increase in VIP immunoreactivity is also seen in this nerve bundle after 4-week PBOO Bar=250µm.

RESULTS 147

Fig 79 Figure shows quantitative comparison of VIP immunoreactivity in DH of L6 and S1

spinal cord before and after 4-week PBOO *p<0.05 when compared with corresponding sham-operated animals.

3.1 Urinary Bladder

Intense NPY immunoreactivity was localized in nerve fibers and nerve trunks coursing through the muscle bundles Some intramural ganglia were also exhibited NPY immunoreactivity (Fig 80)

There was no noticeable change in NPY immunoreactivity at 4 weeks after PBOO when compared with the sham-operated animals

Fig 80 NPY immunoreactivity is localized in

nerve fibers (arrows) and nerve trunks (thick arrows) in muscle layer in sham-operated guinea pig At a higher magnification, NPY immunoreactive nerve fibers appear beaded (B) Arrowheads in A indicate NPY positive intramural ganglion cells bearing long extending processes Bar=125µm (A) and 50µm (B).

3.2 DRG

NPY immunoreactivity was mainly detected in a few small- to medium-sized DRG neurons The frequency of immunopositive neurons was less than 10 per section profile Some immunonegative DRG neurons appeared to be encircled by NPY-IR satellite cells (Fig 81) No apparent change was observed in the number of NPY positive neurons in the DRG after 4-week PBOO

Fig 81 NPY-IR in DRG of normal guinea pig Some small- to medium-sized DRG neurons are

NPY positive (arrows) In some areas, the large NPY negative neurons (thick arrows) are wrapped by NPY positive satellite cells Bar=50µm.

3.3 Spinal Cord

NPY-IR nerve fibers were distributed in the superficial laminae of DH as well

as the area dorsal to the central canal At the sacral level, NPY positive nerve fibers were also found in VH (Fig 82) Rostrally at L6, the NPY-IR fibers disappeared; instead, some neurons in VH were moderately stained (Fig 83)

There was no obvious change in NPY immunoreactivity after 4-week PBOO when compared with the sham-operated animals