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 |