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The objective of this study was to investigate the distribution and chemical coding patterns of nerve fibres supplying the canine urinary bladder before and after botulinum toxin (BTX) injection. The experimental material comprised six bitches. The injection of the BTX into the urinary bladder wall in dogs clearly altered the bladder's innervation pattern, indicating that BTX affects the components of both the sensory and parasympathetic nervous systems, and that degenerative changes are accompanied by restorative processes.
The study was carried out on three 4-month old female pigs. All the animals were deeply anesthetized and transcardially perfused with 4% buffered paraformaldehyde (pH 7.4). Vestibular ganglia (VG) were collected and processed for double-labelling immunofluorescence method. The preparations were examined under the Zeiss LSM 710 confocal microscope equipped with adequate filter blocks. Neurons forming VG were round or oval in shape with a round nucleus in the center. The majority of them (58%) were medium (M) (31-50 μm in diameter) while 28 % and 14% were small (S) (up to 30 μm in diameter) or large (L) (above 50 μm in diameter) in size, respectively. Double-labeling immunofluorescence revealed that VG neurons stained for CGRP (approx. 81%; among them 70.5%, 26.2% and 3.3% were M, S and L in size, respectively), VACHT (57%; 63% M, 24% S, 13% L), Met-Enk (25%; 60% M, 12% S, 28% L), VIP (20%; 88% M, 6% S, L), NPY (15%; 67% M, 20% S, 13% L), GAL (15%; 74% M, 21% S, 5% L), SP (12%; 69% M, 25% S, 6% L) and NOS-positive (12%; 50% S, 50% M). The most abundant populations of intraganglionic nerve fibers were those which stained for CGRP or Met-Enk, whereas only single SP- or NOS-positive nerve terminals were observed.
Single-, double- and triple-labelling immunohistochemistry were applied to study the distribution and neurochemical properties of cholinergic nerve fibres supplying the vas deferens in juvenile and adult pigs. Cholinergic nerves were identified using an antibody against choline acetyltransferase. Immunoblotting was applied to verify the specificity of choline acetyltransferase immunostaining. Western blotting performed on vas deferens tissue homogenates detected single immunoreactive protein with a molecular weight matching this of acetylcholine transferase (71,5 kDa). The majority of choline acetyltransferase-containing nerves innervating the organ are distributed in the lamina propria and coexpress immunoreactivities of up to 4 other biologically active substances including nitric oxide synthase (a marker of nitrergic structures) and neuropeptides, vasoactive intestinal polypeptide, neuropeptide Y and somatostatin. A comparison of data previously collected with the present results reveals that in the pig, neurochemical characteristics of choline acetyltransferase-containing pelvic neurons and nerve fibres supplying the vas deferens are very similar leading to conclusion that cholinergic innervation of the organ largely derives from pelvic ganglia.
The study was performed on six male chinchillas. The animals were anaesthetised with ether and the anaesthesia was deepened with nembuthal injected intraperitoneally. The chinchillas were then transcardially perfused with 0.4 L of 4% buffered paraformaldehyde, and testes, epididymides, and vasa deferentia were collected. The tunica albugínea from one testis from each chinchilla was stained as whole-mount preparation. The tissues were cut into 12 µm-thick cryostat sections, and processed for double-immunofluorescence method. In all organs studied, the most abundant nerve fibres were dopamine ß hydroxylase positive (DßH⁺). Some of them contained neuropeptide Y (NPY). Sporadically NPY-positive-only nerve fibres were found. Single DßH⁺ nerve terminals contained also galanine. Small numbers of the nerve fibres supplying studied organs were stained for substance P (SP) and calitonin gene related peptide (CGRP). Almost all SP⁺ fibres were also CGRP⁺, whereas single CGRP⁺ nerves were SP- immunonegative. Some nerve terminals were immunoreactive to vesicular acetylcholine transporter and vasoactive intestinal peptide. The organs studied were innervated unevenly. The highest density of the nerves was found in the areas of the tunica albuginea adjacent to the mesorchial border of the testis and their number gradually decreased towards the free border of the gonad. None of the vascular tissue of the testicular parenchyma was free of the nerve fibres, except sporadically encountered DßH⁺ nerves which supply seminiferous tubules. Within the head of the epididymis a moderate number of nerve terminals were found, but in the body and tail of the organ the number of nerves gradually increased. The vas deferens was supplied with very numerous nerve fibres. There were no differences in the density of the innervation between the funicular and abdominal part of the vas deferens.
This study describes the expression of DßH as a marker of sympathetic (catecholaminergic) innervation in the vestigial uterus of adult European bison (Eb) bulls. Cryo-sectioned uteri were examined by fluorescent immunohistochemistry with the use of primary rabbit DßH polyclonal antibodies. The DßH-immunocomplexes (antigens/polyclonals) were visualised by secondary donkey antirabbit biotinylated IγG, and then with cyanine (CY™3)-conjugated streptavidin. The DßH immunodetection with CY™3 for the Eb uterine sections was performed in parallel to a positive control (porcine sympathetic paravertebral ganglion), and a negative control (without primary anti-DßH polyclonals). This is the first paper describing the identification of DßH expression within the vestigial uterus of the adult Eb bulls. The distribution pattern of DßH expression localised within immunoreactive (IR) uterine nerve fibres of the bulls resembled that in the control female uterus. The DßH-IR nerve fibres were identified in the entire cross-section of each uterus, with generally higher density in the myometrium than in the endometrium. The ratio of the endometrial/myometrial DßH-expression (percentage of area with IR-signals) was comparable in all males and females (0.78 and 0.64, respectively). However, the DßH-expression area (%) was significantly lower within the male uterine endometrial (P<0.01) and myometrial regions (P<0.001) comparing to the female counterparts. Presumably, the DßH-IR expression within the male Eb uterus is associated with a trophic effect of noradrenaline released by sympathetic nerve fibres influencing nutrient supply of this vestigial organ in the Eb bulls.
To assess the effect of substances inducing mast cell degranulation (substance P and granuliberin R) on the mitotic indices of the gingiva stratified epithelium, basal cells from rats were studied in vivo. Seventy Lewis male rats were used in the study. The rats received injections of either 0.1 ml 0.9% NaCl (10 rats), or substance P (10-4,10-6, 10-8 g/ml) (30 rats), or granuliberin R (10-4,10-6, 10-8 g/ml) (30 rats) into their mandibular gingiva in the vicinity of the right mental foramen. The mitotic index of keratinocytes was established after the kolchicine arrest (2 hours prior to material collection i.p. injection). The number of cells in metaphase was counted on 1000 consecutive basal layer cells after hematoxilin and eosin section staining. Mast cells were revealed using pinocyanol erythrosinate according to Bensley. Numerical density and morphometric features were analyzed. Substance P and granuliberin R injected into the gingiva affect the mast cells and the basal cell proliferation of the gingival epithelium. The diminished mitotic activity of basal layer cells was accompanied by degranulation and/or migration of mast cells under the basal membrane of the epithelium. After administration of high doses of granuloliberin R, mast cells were found in the deep connective tissue alligned towards the epithelium. A neuromediator from the trigeminal nerve (substance P) and substances from mast cells actively interfere in the proliferation of oral keratinocytes and the activity of connective tissue cells.
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