Effect of total or partial uterus extirpation on sympathetic uterus-projecting neurons in porcine inferior mesenteric ganglion. B. Changes in expression of neuropeptide Y,galanin, vasoactive intestinal polypeptide,pituitary adenylate-cyclase activating peptide,somatostatin and substance P

The expression of neuropeptide Y (NPY), galanin (GAL), vasoactive intestinal polypeptide (VIP), pituitary adenylate cyclase-activating peptide (PACAP), somatostatin (SOM) and substance P (SP) was studied in the neurons of the inferior mesenteric ganglion (IMG) projecting to the uterine horn and uterine cervix after uterus extirpation-induced axotomy in sexually immature gilts. The expression was studied with immunohistochemistry, in situ hybridization and RT-PCR. Uterus-projecting neurons were identified by retrograde tracing with Fast Blue (FB). Immunohistochemistry revealed that FB-positive (FB+) uterus-projecting neurons in control animals contained only immunoreactivities to NPY (ca. 50%) and GAL (single neurons). Uterus extirpation increased the occurrence of NPY and GAL in FB+ neurons. No other studied neuropeptides were found in axotomized uterus-projecting neurons. Hybridization in situ revealed the reduction of NPY expression and induction of GAL expression in FB+ neurons. RT-PCR detected induction of GAL expression in the IMG after uterus extirpation. The expression level of NPY and SOM was significant and was not affected by axotomy. The expression level of PACAP was very low and did not differ between IMG of control, partially and totally hysterectomized animals. No VIP and SP expression was detected in all ganglia. The presented data show clear axotomy-related changes in the expression of GAL and NPY in the uterus-projecting neurons of the porcine IMG.     

Chemical Coding of Sensory Neurons Supplying the Hip Joint Capsule in the Sheep

Immunohistochemical properties of nerve fibres supplying the joint capsule were previously described in many mammalian species, but the localization of sensory neurons supplying this structure was studied only in laboratory animals, the rat and rabbit. However, there is no comprehensive data on the chemical coding of sensory neurons projecting to the hip joint capsule (HJC). The aim of this study was to establish immunohistochemical properties of sensory neurons supplying HJC in the sheep. The study was carried out on 10 sheep, weighing about 30–40 kg. The animals were injected with a retrograde neural tracer Fast Blue (FB) into HJC. Sections of the spinal ganglia (SpG) with FB‐positive (FB+) neurons were stained using antibodies against calcitonin gene‐related peptide (CGRP) substance P (SP), pituitary adenylate cyclase‐activating peptide (PACAP), nitric oxide synthase (n‐NOS), neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP), Leu‐5‐enkephalin (Leu‐Enk), galanin (GAL) and vesicular acetylcholine transporter (VACHT). The vast majority of FB+ neurons supplying HJC was found in the ganglia from the 5th lumbar to the 2nd sacral. Immunohistochemistry revealed that most of these neurons were immunoreactive to CGRP or SP (80.7 ± 8.0% or 56.4 ± 4.8%, respectively) and many of them stained for PACAP or GAL (52.9 ± 2.9% or 50.6 ± 19.7%, respectively). Other populations of FB+ neurons were those immunoreactive to n‐NOS (37.8 ± 9.7%), NPY (34.6 ± 6.7%), VIP (28.7 ± 4.8%), Leu‐Enk (27.1 ± 14.6) and VACHT (16.7 ± 9.6).     

Origin and chemical coding of primary afferent neurones supplying the prostate of the dog

Retrograde tracing technique combined with the double-fluorescent immunohistochemistry were used to investigate the distribution and chemical coding of primary afferent neurones supplying the canine prostate. After the injection of Fast Blue (FB) into the prostatic tissue retrogradely-labelled (FB(+)) primary afferent neurones were localized in bilateral L(1)-Ca(1) dorsal root ganglia (DRG). Statistical analysis using anova test showed that there are two major sources of afferent prostate innervation. The vast majority of prostate-supplying primary afferent neurones were located in bilateral L(2)-L(4) DRG (56.9 +/- 0.6%). The second source of the afferent innervation of canine prostate were bilateral S(1)-Ca(1) DRG (40.6 +/- 1.0%). No statistically significant differences were found between average number of FB(+) neurones localized in the left and right DRG (49.5 +/- 1.7 and 50.5 +/- 1.7%, respectively). Immunohistochemistry revealed that FB(+) primary afferent neurones contain several neuropeptides in various combinations. In the prostate-supplying neurones of lumbar and sacro-caudal DRG the immunoreactivity to substance P (SP) and calcitonin gene-related peptide (CGRP) was found most frequently (50 +/- 3.7 and 37.3 +/- 1.9%, respectively). Both in the lumbar and sacro-caudal DRG, considerable population of FB(+) neurones immunoreactive neither to SP nor CGRP were also found (23 +/- 2.6 and 32.8 +/- 2.3%, respectively). In the lumbar DRG 10.7 +/- 1.1% of SP-immunoreactive FB(+) neurones also contained galanin (GAL). In 9.2 +/- 2.2% of the prostate-supplying primary afferent neurones located in the sacro-caudal DRG the co-localization of SP and GAL was also reported. Results of the retrograde tracing experiment demonstrated for the first time sources of afferent innervation of the canine prostate. Double immunohistochemistry revealed that many of the prostate-supplying primary afferent neurones express some of sensory neuropeptides which presumably may be involved in nociception and some pathological processes like inflammation or nerve injury.     

Immunohistochemical Characterization of Sympathetic Chain Ganglia (SChG) Neurons Supplying the Porcine mammary Gland

The aim of this study was to investigate the chemical coding of mammary gland‐projecting SChG neurons using double‐labelling immunohistochemistry. Earlier observation showed that after injection of the retrograde tracer fast blue (FB) into the second, right thoracic mamma, FB+ mammary gland‐projecting neurons were found in Th1‐3, Th9‐14 and L1‐4 right SChG. The greatest number of FB+ nerve cell bodies was observed in Th10 (approx. 843) and Th11 (approx. 567). Neurons projecting to the last right abdominal mamma were found in L1‐4 SChG. The greatest number of FB+ neurons was observed in L2 (approx. 1200). Immunohistochemistry revealed that the vast majority of FB+ mammary‐projecting neurons contained immunoreactivities to TH (96.97%) and/or DßH (95.92%). Many TH/DßH‐positive neurons stained for SOM (41.5%) or NPY (33.2%), and less numerous nerve cells expressed VIP (16.9%). This observation strongly corresponds to the results of previous studies concerning the immunohistochemical characterization of nerve fibres supplying the porcine mammary gland.     

Distribution of neurons supplying the porcine mammary gland

The distribution of neurons projecting to the mammary gland was investigated by using the retrograde tracing method in juvenile pigs (n = 12). Fluorescent retrograde tracer Fast Blue (FB) was injected into the nipple (n = 3) or parenchyma (n = 3) of the second, right thoracic mamma or into the nipple (n = 3) and parenchyma (n = 3) of the last, right abdominal mamma. FB-positive (FB+) mammary gland-projecting neurons were found in some right dorsal root ganglia (DRG) and sympathetic chain ganglia (SChG) only. After injection of the tracer into the second, right thoracic mamma, FB+ neurons were observed in Th9-Th12 DRG but most of them were located in Th11 and Th12 ganglia. As concerns SChG, FB+ neurons were found in Th1-Th4, Th7-Th14 and L1-L4 ganglia. The vast majority of them were located in Th10 and Th11 SChG, which appeared to be the main sources of efferent innervation of this mamma. Neurons projecting to the last right abdominal mamma were found in L1-L3 DRG and L1-L4 SChG but most of them were located in L1-L2 ganglia and L1-L2 ganglia, respectively. This study for the first time has disclosed the localization of neurons supplying the mammary gland in larger domestic animal species, the pig, by using the retrograde tracing method.     

Extrinsic afferents supplying the ovine duodenum and ileum

The study aimed at establishing the distribution of primary sensory neurons by means of retrograde tracers Diamidino Yellow (DY) and Fast Blue (FB) injected into both the sheep duodenum and ileum, respectively. Many DY-labelled cells were found in both the distal vagal ganglia (DVG) and the spinal ganglia (SG) from T9–L3; on the contrary, the majority of the FB-labelled cells were found in the SG. In the SG, a double immunofluorescence stain was used to reveal Nitric Oxide Synthase-Immunoreactivity (NOS-IR) in association with: substance P (SP), calcitonin gene-related peptide (CGRP), neurofilament 200 kDa (NF) and isolectin B₄ (IB4). The labelled neurons, both DY and FB generally ranged in size from medium to large. The majority of the SG duodenal projections were NOS negative; the majority of the SG ileal afferent neurons expressed NOS-IR. Both DY and FB NOS-IR neurons often co-localized IB4, CGRP and SP, but rarely NF.     

Intrinsic innervation of the ileocaecal junction in the horse: Preliminary study

Reason for performing study: In horses, morpho‐functional studies related to the enteric nervous system (ENS) controlling the sphincters are lacking. Objectives: To investigate immunohistochemically the morphology, distribution, density, phenotypes and projections of neurons controlling the ileocaecal junction (ICJ). Methods: Two young horses were anaesthetised and underwent midline laparotomy. The neuronal retrograde fluorescent tracer Fast Blue (FB) was injected into the wall of the ICJ. A post surgical survival time of 30 days was used. Following euthanasia, the ileum and a small portion of caecum were removed. Cryosections were used to investigate the immunoreactivity (IR) of the neurons innervating the ICJ for choline acetyltransferase (ChAT), neuronal nitric oxide synthase (nNOS), substance P (SP), calcitonin gene‐related peptide (CGRP) and neurofilament NF200kDa (NF). Results: Ileal FB‐labelled neurons innervating the ICJ were located in the myenteric plexus (MP) and submucosal plexus (SMP) up to 48 cm and 28 cm, respectively, from the point of the FB injections. Descending MP and SMP neurons were nitrergic (54 ± 11% and 68 ± 4%, respectively), cholinergic (60 ± 19% and 82 ± 11%, respectively), NF‐IR (54 ± 9% and 78 ± 21%, respectively), and SP‐IR (about 20% in both the plexuses). CGRP‐IR was expressed only by SMP descending neurons (45 ± 21%). In both the plexuses descending neurons coexpressing nNOS‐and ChAT‐IR were also observed (25 ± 11% and 61 ± 27%, respectively). Conclusions: The presence of ileal long projecting neurons innervating the ICJ suggests that they are critical for its modulation. Consequently, in bowel diseases in which the resection of the terminal jejunum and proximal ileum are required, it is preferable, whenever possible, to conserve the major portion of the ileum. Potential relevance: The knowledge of the phenotype of ENS neurons of the ileum might be helpful for developing pharmaceutical treatment of the ICJ motility disorders.     

Upregulation of LENK and VIP in paracervical ganglion neurons supplying the urinary bladder of tetrodotoxin- and resiniferatoxin-treated female pigs

Both resiniferatoxin (RTX) and tetrodotoxin (TTX) have been reported to be effective in several clinical trials aiming to cure urinary bladder dysfunction. The goal of this experiment was to study the effect of intravesical administration of RTX and TTX on the chemical coding of paracervical ganglion (PCG) neurons that supply the urinary bladder in pigs. The vasoactive intestinal peptide (VIP) and the opioid family member Leu5-enkephalin (LENK) are both known for their regulatory effects in the function of the porcine genitourinary tract. The PCG neurons innervating the urinary bladder were identified by application of the retrograde tracer Fast Blue (FB), injected into the bladder wall prior to intravesical RTX or TTX administration. Immunocytochemical detection of LENK and VIP expression in the FB-labelled perikarya revealed that in the control group 25.15% of the FB-positive PCG neurons contained LENK, and 9.22% of them expressed VIP. Intravesical infusion of RTX resulted in an increase in the number of LENKIR neurons to 48.19% and VIP-IR perikarya to 11.25%. Optional treatment with TTX induced increase of LENK-IR neurons up to 81.67% and VIP-IR population to 16.46% of the FB-positive PCG cells. The present results show that both neurotoxins affect the chemical coding of PCG nervous cells supplying the porcine urinary bladder and that they stimulate both LENK and VIP expression. Furthermore, the results indicate a possible involvement of LENK and VIP neurons in the mechanisms of action of RTX and TTX in the therapy of overactive bladder disorder.     

Distribution of sympathetic and afferent neurones innervating the submandibular gland in the sheep

The distribution of sympathetic and sensory neurones innervating the submandibular gland (SMG) in sheep was studied using retrograde tracing technique. The retrograde tracer Fast Blue (FB) was unilaterally injected into the SMG in five juvenile male sheep under general anaesthesia. After a 4-week survival period, all the animals were reanaesthetized, perfused transcardially with 4% buffered paraformaldehyde and ganglia, which could be considered as a potential sources of sympathetic, and afferent innervation of the gland were bilaterally collected. The FB-labelled sympathetic neurones were found in the ipsilateral superior and middle cervical ganglion. Many labelled neurones were distributed in the ipsilateral jugular and nodose ganglia of the vagus nerve and smaller numbers of the nerve cells were also found in ipsilateral C1-C3 dorsal root ganglia (DRG). No labelled neurones were observed in the ipsilateral stellate ganglion, trigeminal ganglion, C4-C8 DRG and in all contralateral ganglia. The present study revealed that the majority of sympathetic neurones projecting to the sheep SMG are found in the superior cervical ganglion but some of them are also distributed in the middle cervical ganglion. Most of the afferent neurones are located in the jugular and nodose ganglia of vagus nerve but C1-C3 DRG also comprise some of these nerve cells.     

Immunohistochemical characterization of neurons in the porcine ciliary ganglion

The present study was aimed at disclosing the chemical coding of nerve structures in the porcine ciliary ganglion (CG) using immunohistochemical methods. The substances under investigation included markers of "classical" neurotransmitters, choline acetyltransferase (ChAT), vesicular acetylcholine transporter (VAChT), tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DbetaH) as well as neuropeptides, somatostatin (SOM), galanin (GAL), substance P (SP), calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP) and neuropeptide Y (NPY). Immunoreactivity to ChAT and VAChT was found virtually in all the neuronal somata and in numerous intraganglionic, varicose nerve fibres which often formed basket-like formations around the nerve cell bodies. Many CG neurons contained immunoreactivity for SOM (46%) or GAL (29%). Interestingly, a small number (approx. 1%) of the cholinergic somata stained for TH but not for DbetaH; nevertheless, some extra- and intraganglionic nerve fibres displayed immunoreactivity for DbetaH or TH. The CG perikarya stained neither for vasoactive intestinal polypeptide (VIP) nor for neuropeptide Y (NPY), but some NPY- or VIP-positive nerve terminals were observed within nerve bundles distributed outside the ganglion. SP- and CGRP-immunoreactivity was found in some intraganglionic nerve fibres only. The present study revealed that the porcine CG consists of cholinergic neurons many of which contain SOM and GAL. Thus, it can be assumed that in the pig, these neuropeptides are involved, complementary to acetylocholine, in the parasympathetic postganglionic nerve pathway to structures of the eye including the ciliary and iris sphincter muscles.     

Botulinum toxin type A-induced changes in the chemical coding of dorsal root ganglion neurons supplying the porcine urinary bladder

Botulinum toxin type A (BTX) is a potent neurotoxin, which in recent years has been effectively applied in experimental treatments of many neurogenic disorders of the urinary bladder. BTX is a selective, presynaptically-acting blocking agent of acetylcholine release from nerve terminals what, in turn, leads to the cessation of somatic motor and/or parasympathetic transmission. However, application of this toxin in urological practice is still in the developmental stages and the full mechanism of its action remain elusive. Thus, the present study was aimed at investigating the neurochemical characterization of dorsal root ganglion (DRG) neurons supplying the porcine urinary bladder after BTX treatment. Retrograde tracer Fast Blue (FB) was injected into the urinary bladder wall in six juvenile female pigs and three weeks later, intramural bladder injections of BTX (100 IU per animal) were carried out in all the animals. After a week, DRG from L1 to Cql were harvested from the pigs and neurochemical characterization of FB+ neurons was performed using double- labeling immunofluorescence technique on 10-microm-thick cryostat sections. BTX injections led to a significant decrease in the number of FB+ neurons containing substance P (SP), calcitonin gene-related peptide (CGRP), calbindin (CB), somatostatin (SOM) and neuronal nitric oxide synthase (nNOS) when compared with that found in the healthy animals (19% vs. 45%, 18% vs. 36%, 0.6% vs. 3%, 0.4 vs. 4% and 0.1% vs. 6%, respectively) These data demonstrated that BTX changed the chemical coding of bladder sensory neurons, and therefore this drug should be taken into consideration when it planning experimental therapy of selected neurogenic bladder disorders.     

Conantokin G-induced changes in the chemical coding of dorsal root ganglion neurons supplying the porcine urinary bladder

Conantokin G (CTG), isolated from the venom of the marine cone snail Conus geographus, is an antagonist of N-methyl-d-aspartate receptors (NMDARs), the activation of which, especially those located on the central afferent terminals and dorsal horn neurons, leads to hypersensitivity and pain. Thus, CTG blocking of NMDARs, has an antinociceptive effect, particularly in the case of neurogenic pain treatment. As many urinary bladder disorders are caused by hyperactivity of sensory bladder innervation, it seems useful to estimate the influence of CTG on the plasticity of sensory neurons supplying the organ. Retrograde tracer Fast Blue (FB) was injected into the urinary bladder wall of six juvenile female pigs. Three weeks later, intramural bladder injections of CTG (120 microg per animal) were carried out in all animals. After a week, dorsal root ganglia of interest were harvested from all animals and neurochemical characterization of FB+ neurons was performed using a routine double-immunofluorescence labeling technique on 10-microm-thick cryostat sections. CTG injections led to a significant decrease in the number of FB+ neurons containing substance P (SP), pituitary adenylate cyclase activating polypeptide (PACAP), somatostatin (SOM), calbindin (CB) and nitric oxide synthase (NOS) when compared with healthy animals (20% vs. 45%, 13% vs. 26%, 1.3% vs. 3%, 1.2 vs. 4% and 0.9% vs. 6% respectively) and to an increase in the number of cells immunolabelled for galanin (GAL, 39% vs. 6.5%). These data demonstrated that CTG changed the chemical coding of bladder sensory neurons, thus indicating that CTG could eventually be used in the therapy of selected neurogenic bladder illnesses.     

Immunohistochemical study of the pre- and postnatal innervation of the dog lower urinary tract: morphological aspects at the basis of the consolidation of the micturition reflex

Immunohistochemical studies were performed on male and female bladder and urethra collected from 4 adults dogs and 10 foetal specimens with crown-rump length from 53 to 155 mm (medium-sized breeds, presumptive 38 days of gestation to term). A panel of antisera was tested, including PGP 9.5 to describe the general intramural innervation, ChAT and TH to depict the cholinergic and nor-adrenergic components and NOS1, CGRP, SP, NPY, VIP, SOM, GAL, 5-HT to investigate the possible nitrergic, peptidergic and aminergic ones. A rich cholinergic innervation was present in adult bladder and urethra, along with a lesser number of adrenergic nerves and a small number of nitrergic ones. Either bladder or urethra received numerous CGRP-, SP-, NPY-, VIP-containing nerve fibres which were distributed throughout the muscle layers. All over the lower urinary tract strong to weak ChAT-, CGRP-, SP- and NPY-immunoreactivity was detected in intramural ganglia, in peripheral nerve bundles and around blood vessels. 5-HT-immunoreactive endocrine cells were present in the urethral epithelium. Early foetal organs were supplied only by cholinergic nerve fibres. Few NOS-, CGRP- and SP-ergic components appeared at the end of pregnancy. It can be guessed that sensory mediators such as CGRP and SP increase in postnatal ages while other neuropeptides, such as NPY and VIP, appear only after birth, as the urinary reflex consolidates.     

Immunohistochemical Characterization of Neurones in the Hypoglossal Nucleus of the Pig

With 4 figures and 1 table In this study, the presence of several neurotransmitters and transmitter synthesizing enzymes was studied in hypoglossal nucleus (HN) of the juvenile (4 months old) female pigs (n = 3). Double‐labeling immunofluorescence revealed neurones expressing cholinacetyltranspherase (ChAT), calcitonin gene‐related peptide (CGRP), nitric oxide synthase (NOS), and somatostatin (SOM). Nerve fibers within HN were ChAT, CGRP, NOS, SOM, substance P (SP), Leu‐5‐enkephalin (Leu‐5‐Enk), ß‐dopamine hydroxylase (DßH), neuropeptide Y (NPY) positive. Virtually all the perikarya contained ChAT, whereas CGRP was present in 47% of the neurones. Nerve cell bodies containing NOS or SOM were only occasionally observed. Immunoreactive nerve fibers were found in a close vicinity of the perikarya, often forming baskets around nerve cell bodies. The results obtained were compared with similar data obtained in other species. The presence of immunoreactive structures, origin of the nerve fibers, and functional significance of the findings are discussed.     

Tetrodotoxin induced changes in the chemical coding of dorsal root ganglion neurons supplying the porcine urinary bladder

Tetrodotoxin (TTX) mode of action is based on a blocking of fast sodium channels in nerve cell membrane what, in turn, abolishes the propagation of the action potential along the nerve fibers. TTX is currently used in experimental therapies focused on neoplastic or neurogenic pain, however, as for now there is no data concerning the influence of TTX on dorsal root ganglion (DRG) sensory neurons function. Thus, the present study was aimed at characterization of neurochemical coding of porcine sensory bladder-projecting cells after bladder instillation with TTX. Retrograde tracer Fast Blue (FB) was injected into the urinary bladder wall of six juvenile female pigs and three weeks later bladder instillation with TTX (12 microg per animal) was carried out in all animals. A week later, DRGs of interest were harvested from all animals and the neurochemical characterization of FB+ neurons was performed using routine double-immunofluorescence labeling technique on 10-microm-thick cryostat sections. In TTX-treated animals the number of FB+ cells containing galanin (GAL), nitric oxide synthase (NOS), somatostatin (SOM) and calbindin (CB) was 2.5%, 2%, 0.25% and 0.2%, respectively and that of pituitary adenylate cyclase-activating polypeptide (PACAP)-immunoreactive (IR) cells was 43%. These data when compared with previous reports, demonstrated that TTX profoundly changed the chemical coding of porcine bladder-projecting sensory neurons thus implicating that it may be used in case of hypoactivity of afferent part of reflex arc responsible for transmission of sensory information from the urinary bladder.     

Localisation of recurrent laryngeal nerve motoneurons in the sheep by means of retrograde fluorescent labelling

The purpose of this investigation was to determine the central distribution of the efferent neurons of the recurrent laryngeal nerve (RLN) in the sheep by the use of the retrograde transport of the fluorescent tracer Fast Blue. The distribution of the RLN neurons was also compared with that of the neurons simultaneously labelled by injection of another tracer, Diamidino Yellow dihydrochloride, into the cervical trunk of the vagus nerve (CTV). Injections of the tracer into the CTV resulted in heavy retrograde labelling of neurons in the ipsilateral dorsal motor nucleus of the vagus nerve, in the nucleus ambiguus, in the nucleus retroambigualis and in the reticular formation surrounding the nucleus ambiguus. Following injections of the tracer into the RLN, labelling of neurons was seen over a wide area of the ipsilateral nucleus ambiguus and in the nucleus retroambigualis. Species differences in the distribution of the efferent component of the RLN are discussed, in particular ruminants compared to nonruminants.     

Sources of Sensory Innervation of the Hip Joint Capsule in the Rabbit – A Retrograde Tracing Study

The aim of the study was to investigate the sensory innervation of the hip joint capsule in the rabbit. Individual animals were injected with retrograde fluorescent tracer Fast Blue (FB) into the lateral aspect of the left hip joint capsule (group LAT, n = 5) or into the medial aspect of the hip joint capsule (group MED, n = 5), respectively. FB‐positive (FB+) neurons were found within ipsilateral lumbar (L) and sacral (S) dorsal root ganglia (DRG) from L7 to S2 (group LAT) and from L6 to S4 (group MED). They were round or oval in shape with a diameter of 20–90 μm. The neurons were evenly distributed throughout the ganglia. The average number of FB+ neurons was 16 ± 2.8 and 27.6 ± 3.5 in rabbits from LAT and MED, respectively. The largest average number of FB+ neurons in animals of group LAT was found within the S1 DRG (8 ± 1.7), while S2 ganglion contained the smallest number of the neurons (3.6 ± 1). In the L7 DRG, the average number of FB+ neurons was 6.2 ± 1.6. In rabbits of MED group, the largest number of FB+ neurons was found within the S1 DRG (13.4 ± 4), while the smallest one was found within the S3 ganglion (1.4 ± 0.4). In L6, L7, S2 and S4 ganglia, the number of retrogradely labelled neurons amounted to 1.6 ± 0.5, 4 ± 1.5, 4.4 ± 1.5 and 2.8 ± 1.7, respectively. The data obtained can be very useful for further investigations regarding the efficacy of denervation in the therapy of hip joint disorders in rabbits.     

Effect of total or partial uterus extirpation on uterus-projecting neurons in porcine inferior mesenteric ganglion. C. Changes in expression of apoptosis-associated (Bcl-2 and Bax) and regeneration-associated (GAP-43) proteins

The expression of Bcl-2 and Bax proteins was studied with immunohistochemistry, immuoblotting and RT-PCR in the uterine horn- and uterine cervix-projecting neurons of the inferior mesenteric ganglion (IMG) of the sexually immature gilts after partial or total hysterectomy. Additionally, the expression of regeneration-associated protein GAP-43 was studied in these neurons with immunohistochemistry. The uterus-projecting neurons were identified with retrograde fluorescent tracer Fast Blue (FB). The weak immunoreactivity to Bcl-2 and GAP-43 and moderately intense immunoreactivity to Bax was revealed in all FB+ (FB+) neurons of control and hysterectomized pigs. No difference in the intensity of immunostaining for Bcl-2, Bax and GAP-43 was found between control and hysterectomized gilts. Immunoblotting revealed the presence of Bcl-2 and Bax proteins in IMGs of control and hysterectomized animals and no difference in the band intensities between control and experimental groups was detected. RT-PCR detected weak induction of bcl-2 and bax only in the ganglia of animals which had undergone total hysterectomy. It was found that the axotomy of the uterus-projecting neurons located in the porcine IMG did not change the expression of the studied substances (Bcl-2, Bax and GAP-43) at protein level and only the induction of bcl-2 and bax at the level of RNA was visible.     

Tetrodotoxin- and resiniferatoxin-induced changes in paracervical ganglion ChAT- and nNOS-IR neurons supplying the urinary bladder in female pigs

The aim of the present study was to establish the effect of intravesical administration of resiniferatoxin (RTX) and tetrodotoxin (TTX) on the chemical coding of paracervical ganglion (PCG) neurons supplying the urinary bladder in the pig. In order to identify the PCG neurons innervating the bladder, retrograde tracer Fast Blue was injected into the bladder wall prior to intravesical RTX or TTX administration. Consequent application of immunocytochemical methods revealed that in the control group 76.82% of Fast Blue positive PCG neurons contain nitric oxide synthetase (nNOS), and 66.92% contain acetylcholine transferase (ChAT). Intravesical infusion of RTX resulted in a reduction of the nNOS-IR neurons to 57.74% and ChAT-IR to 57.05%. Alternative administration of TTX induced an increase of nNOS-IR neurons up to 79.29% and a reduction of the ChAT-IR population down to 3.73% of the Fast Blue positive PCG cells. Our data show that both neurotoxins affect the chemical coding of PCG cells supplying the porcine urinary bladder, but the effects of their action are different. Moreover, these results shed light on the possible involvement of NO-ergic and cholinergic neurons in the mechanisms of therapeutic action exerted by RTX and TTX in curing the overactive bladder disorder.     

The influence of resiniferatoxin on the chemical coding of neurons in dorsal root ganglia supplying the urinary bladder in the female pig

Although resiniferatoxin (RTX) becomes more often used in experimental therapies of sensory system disorders, so far there is no data concerning the influence of RTX on the chemical coding of neurons in dorsal root ganglia (DRG) supplying the urinary bladder in the pig, an animal species considered as a reliable animal model for investigation dealing with human lower urinary tract disorders. Retrograde tracer Fast Blue (FB) was injected into the wall of the right half of the urinary bladder in six juvenile female pigs, and three weeks later, bladder instillation of RTX (500 nmol per animal) was carried out in all the animals. After a week, DRGs were harvested from all the pigs and the neurochemical characterization of FB+ neurons was performed using routine single-immunofluorescence labeling technique on 10-microm-thick cryostat sections. RTX instillation resulted in a distinct decrease in the numbers of FB+ cells containing calcitonin gene-related peptide (CGRP), nitric oxide synthase (NOS), somatostatin (SOM) and calbindin (CB) when compared with those found in the healthy animals (18% vs. 36%, 1% vs. 6%, 0.8% vs. 4% and 0.5% vs. 3%, respectively), and an increase in the number of pituitary adenylate cyclase-activating polypeptide (PACAP)- and galanin (GAL)-immunoreactive (IR) nerve cells (51% vs. 26% and 47% vs. 6.5%). The results obtained suggest that RTX could be taken into consideration when the neuroactive agents are planned to be used in experimental therapies of selected neurogenic bladder illnesses.