Immunohistochemical properties of motoneurons supplying the porcine trapezius muscle.

The trapezius muscle (TRAP) belongs to the scapulothoracic group of muscles, which play a crucial role in the integrity and strength of the upper limb, trunk, head, and neck movements and, thus, in maintaining balance. Combined retrograde tracing (using fluorescent tracer Fast Blue, FB) and double-labelling immunohistochemistry were applied to investigate the chemical coding of motoneurons projecting to the porcine TRAP. FB-positive (FB+) motoneurons supplying the cervical (c-TRAP) and thoracic part (th-TRAP) of the right (injected with the tracer) TRAP were located within the IX-th Rexed lamina in the ipsilateral ventral horn of the grey matter of the spinal medulla. Immunohistochemistry revealed that nearly all the neurons were cholinergic in nature [choline acetyltransferase (CHAT)- or vesicular acetylcholine transporter (VACHT)-positive]. Many retrogradelly labelled neurons displayed also immunoreactivity to calcitonin gene-related peptide (CGRP; approximately 68% of FB+ neurons). The smaller number of nerve cells (5%, 3%, 2% or 1%, respectively) stained for nitric oxide synthase (n-NOS), vasoactive intestinal polypeptide (VIP), neuropeptide Y (NPY) and substance P (SP). The retrogradely labelled neurons were closely apposed by nerve fibres expressing immunoreactivity to CHAT, VACHT, CGRP, SP, DßH, VIP, n-NOS, NPY, GAL, Leu-Enk and Met-Enk. Taking into account the clinical relevance of TRAP, the present results may be useful in designing further research aimed at the management of various dysfunctions of the muscle.

Immunohistochemical characterization of nerve elements in porcine intrinsic laryngeal ganglia.

The present study investigated the chemical coding of neurons and nerve fibres in local laryngeal ganglia in pigs (n=5) using double-labelling immunohistochemistry. Virtually all the neurons were cholinergic in nature (ChAT- or VAChT-positive). Only very solitary, small nerve cells (presumably representing interneurons) stained intensely for adrenergic marker, DßH. Many neurons also contained immunoreactivity for NOS (91%), VIP (62.7%), NPY (24.7%), galanin (10%), SP (1.3%) and CGRP (5.3%). No neurons expressing somatostatin or Leu-enkephalin were observed. Nearly all the neuronal somata were densely supplied with varicose cholinergic nerve terminals, which presumably represented preganglionic axons, and some of them were also closely apposed with CGRP- and/or SP-positive varicose nerve endings, which were putative collaterals of extrinsic primary sensory fibres. In conclusion, this study has revealed that intrinsic neurons in the porcine larynx, like in many other mammalian species studied, should be classified as parasympathetic cholinergic neurons expressing biologically active substances, predominantly NOS and VIP. Furthermore, they are likely to receive inputs from not only preganglionic neurons but also primary sensory nerve cells. Finally, it appears that the information on the occurrence of the local laryngeal ganglia should be regularly included in textbooks dealing with the cranial portion of the parasympathetic nervous system in mammals.

The influence of doxazosin on the contractility of the urinary bladder in female pigs with experimentally induced cystitis.

The present in vitro study investigated the influence of doxazosin on the contractility of the urinary bladder in female pigs with experimentally induced cystitis. Fifteen juvenile female piglets (18-20 kg body weight) were randomly assigned into three groups (n=5 animals each): i) control (clinically healthy animals, without doxazosin treatment), ii) animals with induced inflammation of the urinary bladder, but without doxazosin treatment (experimental group I) and iii) animals with inflamed bladder, treated orally with doxazosin (0.1 mg/kg body weight for 30 days; experimental group II). Thereafter, the pigs were sacrificed and strips of the bladder trigone were suspended in organ baths. The tension and amplitude of the smooth muscles was measured before and after exposition to 5-hydroxytryptamine (5-HT; 10-6-10-4 M), acetylocholine (ACh; 10-5-10-3 M) and norepinephrine (NE; 10-9-10-7 M). 5-HT caused an increase in the tension of contractions in all the groups and the amplitude in the experimental groups, however, the effect was higher in the experimental group I than in group II as compared to that found in the pre-treatment period. ACh caused an increase in the tension in the control group and a decrease in the amplitude in both experimental groups; these changes significantly differed between the control and doxazosin-treated group. NE caused a decrease in the tension in both experimental groups and amplitude in all the groups, however, the effect was most strongly expressed in doxazosine-treated group. The present study has revealed that long-term administration of doxazosin causes a desensitization of the detrusor smooth muscle to in vitro applied mediators in the autonomic nervous system.

The influence of resiniferatoxin on the chemical coding of caudal mesenteric ganglion neurons supplying the urinary bladder in the pig.

Resiniferatoxin (RTX) is used as experimental drug therapy for a range of neurogenic urinary bladder disorders. The present study investigated the chemical coding of caudal mesenteric ganglion (CaMG) neurons supplying the porcine urinary bladder after intravesical RTX instillation. The CaMG neurons were visualized with retrograde tracer Fast Blue (FB) and their chemical profile was disclosed with double-labelling immunohistochemistry using antibodies against tyrosine hydroxylase (TH), neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP), somatostatin (SOM), calbindin (CB), galanin (GAL) and neuronal nitric oxide synthase (nNOS). It was found that in both the control (n = 6) and RTX-treated pigs (n = 6), the vast majority (92.3 ± 2.7% and 93.1 ± 1.3%, respectively) of FB-positive (FB+) nerve cells were TH+. Intravesical instillation of RTX caused a decrease in the number of FB+ / TH + neurons immunopositive to NPY (91.0 ± 2.2% in control animals vs. 58.8 ± 5.0% in RTX-treated pigs) or VIP (1.7 ± 0.5% vs. 0%) and an increase in the number of FB+ / TH+ neurons immunoreactive to SOM (3.4 ± 1.5% vs. 20.6 ± 4.3%), CB (1.8 ±0.7% vs. 13.4 ± 2.3%), GAL (1.5 ± 0.6% vs. 7.5 ± 1.0%) or nNOS (0% vs. 10.9 ± 3.4%). The present results suggest that therapeutic effects of RTX on the mammalian urinary bladder can be partly mediated by CaMG neurons.

Overview of the role of vitamins and minerals on the kynurenine pathway in health and disease.

The kynurenine pathway (KP) of L-tryptophan metabolism produces several neuroactive metabolites with an amino acid structure. These metabolites may play an important role in the pathophysiology of irritable bowel syndrome, Alzheimer's disease, Parkinson's disease, Huntington's disease, schizophrenia, AIDS-dementia complex, depression, epilepsy and the aging process. Modulation of the KP through inhibition or stimulation of enzyme synthesis and activity can be an alternative approach to traditional therapy. Furthermore, it may be responsible for the altered functioning of the enteric nervous system and the central nervous system. There is evidence that the KP is sensitive to changes in the concentration of many vitamins and minerals that play a crucial role as coenzymes and cofactors in the de novo synthesis of nicotinamide adenine dinucleotide coenzyme. A reduction in the availability of the active form of vitamin B6 (pyridoxal 5'-phosphate, PLP) is known to affect tryptophan hydroxylase, kynurenine aminotransferase and kynureninase (KYNU). Vitamin B2 deficiencies result in a reduction in the activity of the flavin adenine dinucleotide dependent enzyme, kynurenine 3-monooxygenase. Minerals are also responsible for the proper functioning of enzymes engaged in L-tryptophan metabolism. Mn(2+), Zn(2+), Co(2+) and Cu(2+) influence KYNU activity, and Mg(2+) regulates quinolinate phosphoribosyl transferase. Fe(2+) is responsible for the proper functioning of both indoleamine 2,3-dioxygenase and 3-hydroxy-anthranilic acid dioxygenase. Changes in the concentration of KP metabolites and in enzymatic activity have been found in many pathological states. Therefore, it is justifiable to regulate the concentration of certain kynurenines or enzymes in the KP which may provide a potential therapeutic target for the treatment of various health impairments. This review demonstrates the role of vitamin and mineral activity on the KP, which may have an effect on the proper functioning of the human organism. Surplus administration of vitamins did not elicit any beneficial effects on L-tryptophan metabolism. Whether a mineral surplus influences L-tryptophan metabolism is still not established. It seems that cofactor deficiencies influence the KP far more than surpluses.

Changes in chemical coding of sympathetic chain ganglia (SChG) neurons supplying porcine urinary bladder after botulinum toxin (BTX) treatment.

Botulinum toxin (BTX) is a neurotoxin used in medicine as an effective drug in experimental therapy of neurogenic urinary bladder disorders. We have investigated the influence of BTX on the chemical coding of sympathetic chain ganglia (SChG) neurons supplying the porcine urinary bladder. The toxin was injected into the wall of the bladder. SChG neurons were visualized by a retrograde tracing method with fluorescent tracer fast blue (FB) and their chemical coding was investigated by double-labelling immunohistochemistry with antibodies against dopamine ß-hydroxylase (DßH; a marker of noradrenergic neurons), neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP), somatostatin (SOM), galanin (GAL), Leu(5)-enkephalin (L-ENK) and neuronal nitric oxide synthase (nNOS). In both the control (n = 5) and BTX-treated pigs (n = 5), the vast majority (91 ± 2.3 % and 89.8 ± 2.5 %, respectively) of FB-positive (FB+) nerve cells were DßH+. BTX injections caused a decrease in the number of FB+/DßH+ neurons that were immunopositive to NPY (39.5 ± 4.5 % vs 74.5 ± 11.9 %), VIP (8.9 ± 5.3 % vs 22.3 ± 8.8 %), SOM (5.8 ± 2.3 % vs 17.4 ± 3.7 %) or GAL (0.9 ± 1.2 % vs 5.4 ± 4.4 %) and a distinct increase in the number of FB+/DßH+ neurons that were immunoreactive to L-ENK (3.7 ± 2.9 % vs 1.1 % ± 0.8 %) or nNOS (7.7 ± 3.5 % vs 0.8 ± 0.6 %). Our study provides novel evidence that the therapeutic effects of BTX on the mammalian urinary bladder are partly mediated by SChG neurons.

The influence of botulinum toxin type A (BTX) on the immunohistochemical characteristics of noradrenergic and cholinergic nerve fibers supplying the porcine urinary bladder wall.

Botulinum toxin (BTX) belongs to a family of neurotoxins which strongly influence the function of autonomic neurons supplying the urinary bladder. Accordingly, BTX has been used as an effective drug in experimental therapies of a range of neurogenic bladder disorders. However, there is no detailed information dealing with the influence of BTX on the morphological and chemical properties of nerve fibres supplying the urinary bladder wall. Therefore, the present study investigated, using double-labeling immunohistochemistry, the distribution, relative frequency and chemical coding of cholinergic and noradrenergic nerve fibers supplying the wall of the urinary bladder in normal female pigs (n = 6) and in the pigs (n = 6) after intravesical BTX injections. In the pigs injected with BTX, the number of adrenergic (DbetaH-positive) nerve fibers distributed in the bladder wall (urothelium, submucosa and muscle coat) was distinctly higher while the number of cholinergic (VAChT-positive) nerve terminals was lower than that found in the control animals. Moreover, the injections of BTX resulted in some changes dealing with the chemical coding of the adrenergic nerve fibers. In contrast to the normal pigs, in BTX injected animals the number of DbetaH/NPY- or DbetaH/CGRP-positive axons was higher in the muscle coat, and some fibres distributed in the urothelium and submucosa expressed immunoreactivity to CGRP. The results obtained suggest that the therapeutic effects of BTX on the urinary bladder might be dependent on changes in the distribution and chemical coding of nerve fibers supplying this organ.