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

131 RESULTS 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. RESULTS 132 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 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 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 weeks. Bar=250 m. RESULTS 134 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). RESULTS 136 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 smallor 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). RESULTS 138 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 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) / corresponding sham-operated animals. (for SP) indicate p[...]... 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)  145 RESULTS Fig 75 VIP immunoreactivity in the urinary bladder of guinea pig A shows VIP-IR fibers (arrows) in 4- week... in the guinea pig urinary bladder There is no significant change in expression levels of various cytokines in 4- week PBOO when compared with the sham-operated animals By measuring the OD of each cytokine spot and comparing 4- week sham-operated and PBOO urinary bladder (n =4 for each group), there was no significant change in the respective cytokines except for IL-1 (Fig 85) The intensity of IL-1 increased... seen in VH (thick arrows) Bar=250µm Fig 83 VH neurons slightly stained for NPY in L6 spinal cord of normal guinea pig CC, central canal Bar=250µm RESULTS 150 V EFFECTS OF PBOO ON EXPRESSION OF SOME CYTOKINES IN URINARY BLADDER OF GUINEA PIG Fig 84 shows the representative membrane images for sham-operated and PBOO animals Fig 84 Images show representative membrane images of cytokine antibody array in. .. 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  143 RESULTS Fig 71 SP immunostaining in the lumbosacral spinal... lateral horn (LH) Increase in VIP immunoreactivity is also seen in this nerve bundle after 4- week PBOO Bar=250µm  147 RESULTS Fig 79 Figure shows quantitative comparison of VIP immunoreactivity in DH of L6 and S1 spinal cord before and after 4- week PBOO *p . 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. 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. 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