Figure 1. The distribution and relative frequency of VAChT-IR nerve terminals in the control (contr.; A–C), RTX-treated (RTX; D–F), or TTX-treated (TTX; G–I) pigs; muscle layer (mL), blood vessel (bv), submucosa (s), urothelium (u); 20×.
Figure 1. The distribution and relative frequency of VAChT-IR nerve terminals in the control (contr.; A–C), RTX-treated (RTX; D–F), or TTX-treated (TTX; G–I) pigs; muscle layer (mL), blood vessel (bv), submucosa (s), urothelium (u); 20×.
Figure 2. Distribution of VAChT-(red; labelled with CY3) and calcitonin gene-related peptide- (CGRP)- (A–C), neuronal nitric oxide synthase- (nNOS)- (D–F), neuropeptide Y- (NPY)- (G–I), somatostatin- (SOM)- (J–L) or vasoactive intestinal polypeptide- (VIP)- (M–O) positive (green; labelled with fluorescein isothiocyanate (FITC)) nerve fibers in the urinary bladder wall in the normal (A,D,G,J,M), RTX-treated (B,E,H,K,N), or TTX-treated (C,F,I,L,O) pigs. Red and green channels were digitally superimposed. Double-labelled fibers are yellow to orange, and most of them are indicated with arrows; muscle layer (mL), blood vessel (bv), submucosa (s), urothelium (u); 20×.
Figure 2. Distribution of VAChT-(red; labelled with CY3) and calcitonin gene-related peptide- (CGRP)- (A–C), neuronal nitric oxide synthase- (nNOS)- (D–F), neuropeptide Y- (NPY)- (G–I), somatostatin- (SOM)- (J–L) or vasoactive intestinal polypeptide- (VIP)- (M–O) positive (green; labelled with fluorescein isothiocyanate (FITC)) nerve fibers in the urinary bladder wall in the normal (A,D,G,J,M), RTX-treated (B,E,H,K,N), or TTX-treated (C,F,I,L,O) pigs. Red and green channels were digitally superimposed. Double-labelled fibers are yellow to orange, and most of them are indicated with arrows; muscle layer (mL), blood vessel (bv), submucosa (s), urothelium (u); 20×.
Figure 3. The distribution and relative frequency of DβH-immunoreactive nerve fibers in control (contr.; A–C), RTX-treated (RTX; D–F), or TTX-treated (TTX; G–I) pigs; muscle layer (mL), blood vessel (bv), submucosa (s), urothelium (u); 20×.
Figure 3. The distribution and relative frequency of DβH-immunoreactive nerve fibers in control (contr.; A–C), RTX-treated (RTX; D–F), or TTX-treated (TTX; G–I) pigs; muscle layer (mL), blood vessel (bv), submucosa (s), urothelium (u); 20×.
Figure 4. Distribution of DβH-(green; labelled with FITC) and GAL- (A–C), L-ENK- (D–F), nNOS- (G–I), NPY- (J–L) or SOM- (M–O) positive (red; labelled with CY3) nerve fibers in the urinary bladder wall in the normal (A,D,G,J,M), RTX-treated (B,E,H,K,N), or TTX-treated (C,F,I,L,O) pigs. Red and green channels were digitally superimposed. Double-labelled fibers are yellow to orange and most of them are indicated with arrows; muscle layer (mL), blood vessel (bv), submucosa (s), urothelium (u); 20× (A–O); 40× (I).
Figure 4. Distribution of DβH-(green; labelled with FITC) and GAL- (A–C), L-ENK- (D–F), nNOS- (G–I), NPY- (J–L) or SOM- (M–O) positive (red; labelled with CY3) nerve fibers in the urinary bladder wall in the normal (A,D,G,J,M), RTX-treated (B,E,H,K,N), or TTX-treated (C,F,I,L,O) pigs. Red and green channels were digitally superimposed. Double-labelled fibers are yellow to orange and most of them are indicated with arrows; muscle layer (mL), blood vessel (bv), submucosa (s), urothelium (u); 20× (A–O); 40× (I).
Table 1. The distribution and relative frequency of vesicular acetylcholine transporter-immunoreactive (VAChT-IR) nerve fibers supplying the porcine urinary bladder wall. RTX = resiniferatoxin (RTX); TTX = tetrodotoxin; − nerve fibers not found; +/− single fibers; + few fibers; ++ moderate number of fibers; +++ many fibers; ++++ a very dense meshwork of fibers; ↓ a decrease in the nerve fibers density.
Table 1. The distribution and relative frequency of vesicular acetylcholine transporter-immunoreactive (VAChT-IR) nerve fibers supplying the porcine urinary bladder wall. RTX = resiniferatoxin (RTX); TTX = tetrodotoxin; − nerve fibers not found; +/− single fibers; + few fibers; ++ moderate number of fibers; +++ many fibers; ++++ a very dense meshwork of fibers; ↓ a decrease in the nerve fibers density.
Part of the Urinary Bladder Wall |
Control Pigs |
RTX-Treated Pigs |
TTX-Treated Pigs |
Muscle layer |
++++ |
++ ↓ |
++ ↓ |
Submucosa |
++ |
++ |
+/− ↓ |
Urothelium |
+ |
+ |
− ↓ |
Blood vessels |
+++ |
+ ↓ |
+/− ↓ |
Table 2. The degree of colocalization of VAChT with other immunoreactive substances within the nerve fibers supplying the urinary bladder wall. mL—muscle layer; bv—blood vessels; s – submucosa; u – urothelium; − nerve fibers not found; +/− single fibers; + few fibers; ++ moderate number of fibers; +++ many fibers; ++++ a very dense meshwork of fibers. GAL= galanin; L-ENK = Leu5–enkephalin; PACAP = pituitary adenylate cyclase-activating polypeptide; SP = substance P; ↓ a decrease in the nerve fibers density.
Table 2. The degree of colocalization of VAChT with other immunoreactive substances within the nerve fibers supplying the urinary bladder wall. mL—muscle layer; bv—blood vessels; s – submucosa; u – urothelium; − nerve fibers not found; +/− single fibers; + few fibers; ++ moderate number of fibers; +++ many fibers; ++++ a very dense meshwork of fibers. GAL= galanin; L-ENK = Leu5–enkephalin; PACAP = pituitary adenylate cyclase-activating polypeptide; SP = substance P; ↓ a decrease in the nerve fibers density.
Substances |
Control Pigs |
RTX-Treated Pigs |
TTX-Treated Pigs |
mL |
bv |
s |
u |
mL |
bv |
s |
u |
mL |
bv |
s |
u |
VAChT/CGRP |
+ |
+ |
+ |
+ |
+/− ↓ |
- ↓ |
− ↓ |
− ↓ |
+ |
− ↓ |
− ↓ |
− ↓ |
VAChT/GAL |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
VAChT/L-ENK |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
VAChT/nNOS |
+++ |
+ |
+ |
- |
+ ↓ |
+/− ↓ |
- |
- |
− ↓ |
− ↓ |
− ↓ |
- |
VAChT/NPY |
+++ |
+ |
- |
- |
++ ↓ |
+/− ↓ |
- |
- |
+++ |
+ |
- |
- |
VAChT/PACAP |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
VAChT/SOM |
++++ |
++++ |
++++ |
+ |
++ ↓ |
+/− ↓ |
+ ↓ |
- |
++ ↓ |
+ ↓ |
++ ↓ |
+/− ↓ |
VAChT/SP |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
VAChT/VIP |
+ |
+ |
++ |
+ |
+/− ↓ |
− ↓ |
− ↓ |
− ↓ |
+ |
− ↓ |
+ ↓ |
− ↓ |
Table 3. The distribution and relative frequency of dopamine β-hydroxylase-immunoreactive (DβH-IR) nerve fibers supplying the porcine urinary bladder wall; − nerve fibers not found; +/− single fibers; + few fibers; ++ moderate number of fibers; +++ many fibers; ++++ a very dense meshwork of fibers; ↓ a decrease in the nerve fibers density; ↑ an increase in the nerve fibers density.
Table 3. The distribution and relative frequency of dopamine β-hydroxylase-immunoreactive (DβH-IR) nerve fibers supplying the porcine urinary bladder wall; − nerve fibers not found; +/− single fibers; + few fibers; ++ moderate number of fibers; +++ many fibers; ++++ a very dense meshwork of fibers; ↓ a decrease in the nerve fibers density; ↑ an increase in the nerve fibers density.
Part of the Urinary Bladder Wall |
Control Pigs |
RTX-Treated Pigs |
TTX-Treated Pigs |
Muscle layer |
+ |
+ |
++ ↑ |
Submucosa |
++ |
+ ↓ |
+++ ↑ |
Urothelium |
+/− |
− ↓ |
+ ↑ |
Blood vessels |
++++ |
++++ |
++++ |
Table 4. The degree of colocalization of DβH with other immunoreactive substances within the nerve fibers supplying the urinary bladder wall. Muscle layer (mL); blood vessels (bv); submucosa (s); urothelium (u); − nerve fibers not found; +/− single fibers; + few fibers; ++ moderate number of fibers; +++ many fibers; ++++ a very dense meshwork of fibers. ↓ a decrease in the nerve fibers density; ↑ an increase in the nerve fibers density.
Table 4. The degree of colocalization of DβH with other immunoreactive substances within the nerve fibers supplying the urinary bladder wall. Muscle layer (mL); blood vessels (bv); submucosa (s); urothelium (u); − nerve fibers not found; +/− single fibers; + few fibers; ++ moderate number of fibers; +++ many fibers; ++++ a very dense meshwork of fibers. ↓ a decrease in the nerve fibers density; ↑ an increase in the nerve fibers density.
Substances |
Control Pigs |
RTX-Treated Pigs |
TTX-Treated Pigs |
mL |
bv |
s |
u |
mL |
bv |
s |
u |
mL |
bv |
s |
u |
DβH/CGRP |
+ |
- |
- |
- |
+ |
- |
- |
- |
+ |
- |
- |
- |
DβH/GAL |
- |
- |
- |
- |
+ ↑ |
+ ↑ |
- |
- |
+ ↑ |
- |
- |
- |
DβH/L-ENK |
++ |
+/− |
+ |
+ |
++ |
+/− |
+ |
+ |
+++ ↑ |
++ ↑ |
+ |
+ |
DβH/nNOS |
- |
- |
- |
- |
- |
- |
+ ↑ |
+ ↑ |
- |
- |
++ ↑ |
++ ↑ |
DβH/NPY |
++++ |
++++ |
+ |
+ |
++ ↓ |
+ ↓ |
+/− ↓ |
− ↓ |
++ ↓ |
++ ↓ |
+/− ↓ |
− ↓ |
DβH/PACAP |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
DβH/SOM |
++ |
+/− |
+ |
+ |
++ |
+/− |
+ |
+ |
+++ ↑ |
+ ↑ |
+ |
+ |
DβH/SP |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
DβH/VIP |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
Table 5. List of primary antisera and secondary reagents used in the study (CGRP = calcitonin gene-related peptide, DβH = dopamine β-hydroxylase, GAL = galanin, L-ENK = Leu5–enkephalin, nNOS = neuronal nitric oxide synthase, NPY = neuropeptide Y, PACAP = pituitary adenylate cyclase-activating polypeptide, SOM = somatostatin, SP = substance P, VAChT = vesicular acetylcholine transporter, VIP = vasoactive intestinal polypeptide, FITC = fluorescein isothiocyanate.
Table 5. List of primary antisera and secondary reagents used in the study (CGRP = calcitonin gene-related peptide, DβH = dopamine β-hydroxylase, GAL = galanin, L-ENK = Leu5–enkephalin, nNOS = neuronal nitric oxide synthase, NPY = neuropeptide Y, PACAP = pituitary adenylate cyclase-activating polypeptide, SOM = somatostatin, SP = substance P, VAChT = vesicular acetylcholine transporter, VIP = vasoactive intestinal polypeptide, FITC = fluorescein isothiocyanate.
Antigen |
Code |
Dilution |
Species |
Supplier |
Primary antibodies |
CGRP |
T-5027 |
1:400 |
Guinea pig |
Peninsula; San Carlos; CA; USA |
AB5920 |
1:8000 |
Rabbit |
Millipore; Temecula; CA; USA |
DβH |
MAB 308 |
1:300 |
Mouse |
Millipore; Temecula; CA; USA |
D9010-07A.50 |
1:4000 |
Rabbit |
Biomol; Hamburg; Germany |
GAL |
T-5036 |
1:1000 |
Guinea pig |
Peninsula; San Carlos; CA; USA |
AB 5909 |
1:4000 |
Rabbit |
Millipore; Temecula; CA; USA |
L-ENK |
4140-0355 |
1:800 |
Mouse |
Bio-Rad; Kidlington; UK |
AB5024 |
1:600 |
Rabbit |
Merck; Darmstadt; Germany |
nNOS |
N2280 |
1:400 |
Mouse |
Sigma; MSU; USA |
AB 5380 |
1:17000 |
Rabbit |
Millipore; Temecula; CA; USA |
NPY |
NA1233 |
1:8000 |
Rabbit |
Enzo Life Sciences; Farmingdale; NY; USA |
sc-133080 |
1:100 |
Mouse |
Santa Cruz Biotechnology; TX; USA |
PACAP |
T-5039 |
1:300 |
Guinea pig |
Peninsula; San Carlos; CA; USA |
T-4465 |
1:20000 |
Rabbit |
Peninsula; San Carlos; CA; USA |
SOM |
11180 |
1:30 |
Rabbit |
Icn-Cappel; Aurora; OH; USA |
T-1608 |
1:30 |
Rat |
Peninsula; San Carlos; CA; USA |
SP |
8450-0505 |
1:100 |
Rat |
Bio-Rad; Kidlington; UK |
VAChT |
H-V006 |
1:6000 |
Rabbit |
Phoenix Pharmaceuticals Inc; Burlingame; CA; USA |
VIP |
VA 1285 |
1:6000 |
Rabbit |
Enzo Life Sciences; Farmingdale; NY; USA |
T-5030 |
1:1000 |
Guinea pig |
Peninsula; San Carlos; CA; USA |
Secondary reagents |
Biotinylated anti-rabbit immunoglobulins |
E 0432 |
1:800 |
Goat |
Dako; Hamburg; Germany |
CY3-conjugated streptavidin |
711-165-152 |
1:8000 |
- |
Jackson I.R.; West Grove; PA; USA |
FITC-conjugated anti-mouse IgG |
715-096-151 |
1:400 |
Donkey |
Jackson I.R.; West Grove; PA; USA |
FITC-conjugated anti-rat IgG |
712-095-153 |
1:400 |
Donkey |
Jackson I.R.; West Grove; PA; USA |
FITC-conjugated anti-guinea pig IgG |
706-095-148 |
1:600 |
Donkey |
Jackson I.R.; West Grove; PA; USA |
Table 6. List of antigens used in pre-absorption test.
Table 6. List of antigens used in pre-absorption test.
Antigens Used in Pre-Absorption Test |
CGRP |
C0292 |
Sigma; MSU; USA |
DβH-blocking peptide |
MBS9218238 |
MyBioSource; CA; USA |
GAL |
G5773 |
Sigma; MSU; USA |
L-ENK |
ab142314 |
Abcam; UK |
nNOS |
N3033 |
Sigma; MSU; USA |
NPY |
N3266 |
Sigma; MSU; USA |
PACAP |
A9808 |
Sigma; MSU; USA |
SOM |
S9129 |
Sigma; MSU; USA |
SP |
S6883 |
Sigma; MSU; USA |
VAChT |
V007 |
Phoenix Pharmaceuticals Inc; CA; USA |
VIP |
V6130 |
Sigma; MSU; USA |