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

RESULTS 116 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. RESULTS 117 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. RESULTS 118 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. RESULTS 119 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. RESULTS 120 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. RESULTS 121 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 RESULTS 122 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 RESULTS 123 (Figs. 42, 43B). nNOS immunoreactive precipitates were also found in satellite cells (Figs. 43 A, C-D). 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). RESULTS 124 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). RESULTS 125 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). RESULTS 126 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. RESULTS 127 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). RESULTS 128 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. RESULTS 129 3.2.Spinal Cord The number of immunopositive cells in the DH was significantly increased at weeks after PBOO, but decreased marginally at 12 weeks when compared to that at 4-week (Fig. 49). Results of cell counting are shown in Fig. 50. In addition to the increased number of nNOS positive neurons in DH, the bundle of nNOS-IR nerve fibers running laterally at sacral segments also showed enhanced immunoreactivity at weeks after PBOO, when compared with normal (Fig. 51). Fig. 49. nNOS immunostaining in the sacral spinal cord(S1-2). A: normal control; B: 4-week PBOO; C: 12-week PBOO. Bar=250µm. RESULTS 130 Fig. 50. Graph shows cell counting result of nNOS positive neurons in DH. Values represent average cell number per section (mean±SD). *p[...]... 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) RESULTS 128 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... 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... compared to that at 4-week (Fig 49) Results of cell counting are shown in Fig 50 In addition to the increased number of nNOS positive neurons in DH, the bundle of nNOS-IR nerve fibers running laterally at sacral segments also showed enhanced immunoreactivity at 4 weeks after PBOO, when compared with normal (Fig 51) Fig 49 nNOS immunostaining in the sacral spinal cord(S1-2) A: normal control; B: 4-week... 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 RESULTS 127 Fig 47 NADPH-d staining in the spinal cord at the sacral level (S1-2) As in nNOS immunostaining, NADPH-d positive... 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 RESULTS 129 3. 2.Spinal Cord The number of immunopositive cells in the DH was significantly increased at 4 weeks after PBOO, but decreased marginally at 12... 12-week PBOO Bar=250µm RESULTS 130 Fig 50 Graph shows cell counting result of nNOS positive neurons in DH Values represent average cell number per section (mean±SD) *p . 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. section profile. Bar=250µm in A; =125µm in B and =50µm in C. RESULTS 120 Fig. 35 . nNOS immunofluorescence is localized in a neuron (arrow) in the S1 DRG of a normal guinea pig. Bar=125µm in A, and. 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