Non adrenergic non cholinergic innervation of lower urinary tract after partial bladder outlet obstruction in guinea pig 3

Light Microscopic Observations In normal control guinea pigs, the majority of the nNOS-IR neurons were small cells measuring an average diameter 25µm Figs.. NADPH-d staining showed that

III EFFECTS OF PBOO ON nNOS EXPRESSION IN DRG AND SPINAL CORD OF GUINEA PIG

1 Retrograde Axonal Tracing Study to Localize the Bladder Afferent Neurons

in DRG

Following injection of FG into the bladder wall of normal guinea pig, the fluorescent dye was detected in the bilateral DRG neurons The FG-labeled cells were randomly distributed in the ganglia (Fig 32) The majority of the FG-labeled neurons were found bilaterally in the DRG at levels L6 and S1-S3 segments A few labeled cells were detected in DRG as far as at L1 level rostrally (Fig 33)

Fig 32 FG-labeled neurons in DRG after injection of FG into urinary bladder wall of normal

guinea pig A, B and C show the same ganglion (S2) at different magnification: 4x 10x and 20x objective, respectively Arrows indicate labeled neurons Note the elliptical shape of labeled neurons Bar=500µm, 250µm and 125µm, in A, B and C, respectively.

Fig 33 Majority of FG-labeled neurons are distributed in L6, S1-S3 DRG A, B and C show

representative images of S1, S2 and S3 DRG D, E and F show representative images of L1, L2 and L3 DRG A few labeled neurons are detected in L1 DRG Bar=250µm.

2 Distribution of nNOS Immunoreactivity

2.1 DRG

2.1.1 Light Microscopic Observations

In normal control guinea pigs, the majority of the nNOS-IR neurons were small cells measuring an average diameter 25µm (Figs 34 and 35) In general, the number

of nNOS positive neurons in one DRG section was less than 10 (Fig 34) Bundles of nNOS-IR neurites appeared to course between the neuronal cell bodies and sometimes, encircled them (Fig 36)

The majority of DRG neurons were large- and medium-sized, measuring about 70µm and 50µm in diameter, respectively These neurons were mostly nNOS immunonegative neurons Delineating the nNOS negative soma were nNOS positive structures forming a thin ring Immunofluorescence labeling revealed that they were either derived from the pericellular nerve fiber network (Fig 36) or associated satellite cells (Figs 37 and 38)

NADPH-d staining showed that a large number of DRG neurons were NADPH-d positive The NADPH-d positive neurons were more numerous than the nNOS-IR neurons Most of the intensely stained neurons were small cells (Fig 39),

a feature that is consistent with the nNOS immunohistochemical staining

Fig 34 Representative images of nNOS positive neurons (arrows) in L6 DRG of normal guinea

pig at different magnifications The nNOS staining is localized primarily in small neurons The number of nNOs positive neurons is in general less than 10 neurons per section profile Bar=250µm in A; =125µm in B and =50µm in C.

Fig 35 nNOS immunofluorescence is localized in a neuron (arrow) in the S1 DRG of a normal

guinea pig Bar=125µm in A, and =50µm in B.

Fig 36 nNOS immunofluorescent nerve bundles (arrow) traversing through the S1 DRG

ganglion Bar=50µm.

Fig 37 Some satellite cells wrapping the DRG (L6) neurons are also immunoreactive for nNOS

(arrows) Arrowheads indicate nNOS positive neurons Bar=50µm.

Fig 38 nNOS immunofluorescence is detected in satellite cells (arrows) wrapping the large

nNOS negative neurons Arrowhead indicates small nNOs positive neuron Bar=50µm (A) and 100µm (B).

Fig 39 NADPH-d reactivity in DRG (L6) of normal guinea pig Compared to nNOS

immunohistochemical staining, there are more NADPH-d positive neurons than nNOS positive neurons The small cells are more intensely stained than large neurons Arrows indicate intensely stained small neurons Arrowhead indicates an unstained large neuron Bar=250µm.

2.1.2 Electron Microscopic Observations

2.1.2.1 Conventional Electron Microscopic Observations

Normal DRG neurons displayed common neuronal features, e.g a large, round, pale nucleus (Fig 40) The cytoplasm contained abundant organelles (Fig 40) The neuronal perikarya were surrounded by satellite cells (Fig 40) which had a smaller

and elongated nucleus showing dense chromatin masses Myelinated and unmyelinated nerves fibers consisting of a varying number of axons were found in the connective tissues around the neurons (Figs 40 and 41)

Fig 40 Electron micrograph showing two typical neurons N1, N2 in normal guinea pig L6 DRG.

The soma of the neuron is enwrapped by slender processes (arrows) of satellite cells (S) Both myelinated (M) and unmyelinated (arrowheads) nerve fibers are seen near the neuronal somata Bar=2µm.

Fig 41 Electron micrographs show myelinated (arrows) and unmyelinated (arrowheads) nerve

fibers in normal guinea pig L6 DRG SW in B indicates Schwann cell Bar=0.5µm (A) and 1µm (B).

2.1.2.2 Immunoelectron Microscopic Observations

With nNOS immunohistochemical staining, the reaction product was deposited

on the membranes of various sub-cellular organelles, especially the mitochondria

Fig 42 Electron micrographs showing nNOS positive neurons in normal guinea pig S1 DRG.

The immuno-precipitates are mainly distributed on the membranes of organelles, especially the mitochondria (arrows) Arrowhead in B (higher magnification) indicates negative mitochondria among the nNOS-IR mitochondria N, nucleus Bar=1µm (A) and 0.2µm (B).

Fig 43 Electron micrographs showing nNOS positive satellite cells wrapping nNOS negative

neurons in normal guinea pig S1 DRG (A, C-D) B shows a nNOS positive neuron wrapped by

an nNOS negative satellite cell The nNOS-IR products (arrows) in satellite cells are distributed

in mitochondria or randomly in the cytoplasm N: neuron; S: satellite cell; NN: neuronal nucleus; SN: satellite cell nucleus Bar=0.5µm (A, B); 1µm (C) and 2µm (D).

2.2.Spinal Cord

nNOS immunoreactivity was detected in neurons and nerve fibers in the superficial area of the DH and in neurons around the central canal (Fig 44) A few nNOS-IR neurons were scattered in the deeper laminae and VH Small, round or oval nNOS positive cells occurred mostly in the superficial layers, whereas larger, polymorphic cells with long extending processes were in the deeper laminae nNOS-IR nerve fibers were also found running as a bundle along the lateral edge of the DH to the region of the lateral horn in the sacral spinal cord (Figs 44, 45)

In the thoracolumbar spinal cord, nNOS immunoreactivity was also present in the sympathetic preganglionic neurons located in the ILN In addition, nNOS positive nerve fibers were also detected in the area of dorsal commissure (Fig 46)

On the other hand, nNOS immunoreactivity was absent in the region of lateral horn

in the sacral spinal cord (refer to Fig 44A)

The distribution pattern of NADPH-d reactivity was comparable to that of nNOS immunoreactivity (Fig 47)

Fig 44 Localization of nNOS immunoreactivity in the spinal cord at sacral level (S1-2) of

normal guinea pig Bar=250µm (A); 125µm (B) and 50µm (C).

Fig 45 nNOS immunoreactivity in the lumbosacral spinal cord (S1-2) in normal guinea pig A

shows nNOS positive neurons around the central canal (CC) Note the long extending processes

of the stained cells B shows nNOS positive nerve fibers (arrows) extending along the lateral edge of the dorsal horn (DH) to the region of the lateral horn (LH) The nNOS-IR nerve fibers are especially conspicuous in the S1-S3 segments C shows a few lightly stained nNOS neurons (arrows) scattered in the ventral horn Bar=50µm (A and C); 100µm (B).

Fig 46 L1 segment of spinal cord Note nNOS positive neurons are present in the ILN; also,

nNOS-IR nerve fibers are detected in the dorsal commissure (arrow) Bar=250µm.

Fig 47 NADPH-d staining in the spinal cord at the sacral level (S1-2) As in nNOS

immunostaining, NADPH-d positive neurons are detected in DH, and around the central canal (CC) In addition, NADPH-d positive fibers are present in the superficial layers of DH and the lateral edge of the DH extending into the region of the lateral horn (LH) Bar=250µm (A); 125µm (B) and 50µm (C).

3 Changes in nNOS Expression in the DRG and Spinal Cord after PBOO 3.1 DRG

There was no noticeable change in nNOS positive neurons in the lumbosacral DRG after PBOO when compared with the control (Fig 48)

Fig 48 DRG at sacral level (S1-2) of normal (A), 4-week PBOO (B) and 12-week PBOO (C).

Note nNOS immunoreactivity in DRG between the control and those after PBOO is comparable Bar=250µm.

Fig 49 nNOS immunostaining in the sacral spinal cord(S1-2) A: normal control; B: 4-week

PBOO; C: 12-week PBOO Bar=250µm.

Fig 50 Graph shows cell counting result of nNOS positive neurons in DH Values represent

average cell number per section (mean±SD) *p<0.05 when compared to control.

Fig 51 At 4 weeks after PBOO, the expression of nNOS in the nerve fibers (arrows) extending

along the lateral edge of the DH to the region of the lateral horn is markedly increased A: normal; B: 4-week PBOO Bar=50µm.