Effect of different atmospheric and subatmospheric cooking techniques on physical and chemical qualitative properties of pork loin.

In this paper, the effects of four cooking procedures were evaluated, two occurring in atmospheric (in ventilated and steam oven) and two in subatmospheric (vacuum and sous vide cooking) conditions on pork Longissimus lumborum. The main objective of the study was to compare and evaluate the physical and chemical characteristics. Samples were cooked in four independent trials namely Oven (O), Steaming (ST), Vacuum Cooking (VC) and Sous Vide (SV). The analyses included temperature, cooking effect, percentage weight loss, texture (cutting and double compression tests), colour (superficially and inside the sample), microstructure (optical microscopy) and fibres shortening analysis. To assess cooking effects on significant nutritional constituents, the fatty acid composition and the content of B vitamins were analysed. Volatile profiles of samples were also compared using solid-phase microextraction. SV cooking resulted in the less favourable meat texture, presenting the highest hardness and chewiness. Moreover, high hardness values measured on SV samples is also related to the high weight loss. The technique of oven cooking (O) demonstrated superior results in terms of mechanical properties, which are closely associated with the cooking values. Specifically, the cook value C0 was significantly higher in the case of oven cooking compared to SV, VC, and ST. Mild temperature conditions and cooking times of the four considered cooking techniques did not induce significant variations in the fatty acid composition and volatile profile. Conversely, SV and VC allowed the highest amount of vitamin B retention in cooked meat. This work suggests that some differences emerged on the effects due to sub-atmospheric and atmospheric cooking compared to traditional ones.

Preterm rabbit-derived Precision Cut Lung Slices as alternative model of bronchopulmonary dysplasia in preclinical study: a morphological fine-tuning approach.

Bronchopulmonary dysplasia (BPD) is the most common complication of preterm delivery, with significant morbidity and mortality in a neonatal intensive care setting. Research in this field aims to identify the mechanisms of late lung development with possible therapeutic targets and the improvement of medical management. Rabbits represent a suitable lab preclinical tool for mimicking the clinical BPD phenotype. Rabbits are born at term in the alveolar phase as occurs in large animals and humans and in addition, they can be delivered prematurely in contrast to mice and rats. Continuous exposure to high oxygen concentration (95% O2) for 7 days induces functional and morphological lung changes in preterm rabbits that resemble those observed in BPD-affected babies. The preclinical research pays great attention to optimize the experimental procedures, reduce the number of animals used in experiments and, where possible, replace animal models with alternative assays, following the principle of the 3 Rs (Replace, Reduce and Refine). The use of in vitro assays based on the ex vivo culture of Precision Cut Lung Slices (PCLS) goes in this direction, representing a good compromise between controlled and flexible in vitro models and the more physiologically relevant in vivo ones. This work aims to set up morphological analyses to be applied in preclinical tests using preterm rabbits derived PCLS, cultured up to 7 days in different oxygen conditions, as a model. After a preliminary optimization of both lung preparation and histological processing methods of the lung slices of 300 µm, the morphological analysis was conducted evaluating a series of histomorphometric parameters derived from those widely used to follow the phases of lung development and its alterations in vivo. Our histomorphometric results demonstrated that the greatest differences from pseudo-normoxia and hyperoxia exposed samples at day 0, used as starting points to compare changes due to treatments and time, are detectable after 4 days of in vitro culture, representing the most suitable time point for analysis in preclinical screening. The combination of parameters suitable for evaluating PCLS morphology in vitro resulted to be Tissue Density and Septal Thickness. Shape Factor and Roughness, evaluated to highlight the increasing complexity of the airspaces, due to the formation of septal crests, gave useful information, however, without significant differences up to day 4. Other parameters like Mean Linear Intercept and Septal Density did not allow to highlight significant differences between different oxygen conditions and time points. Instead, Radial Alveolar Count, could not be applied to PCLS, due to the tissue changes following agar infusion and culture conditions.

Synthetic recovery of impulse propagation in myocardial infarction via silicon carbide semiconductive nanowires.

Myocardial infarction causes 7.3 million deaths worldwide, mostly for fibrillation that electrically originates from the damaged areas of the left ventricle. Conventional cardiac bypass graft and percutaneous coronary interventions allow reperfusion of the downstream tissue but do not counteract the bioelectrical alteration originated from the infarct area. Genetic, cellular, and tissue engineering therapies are promising avenues but require days/months for permitting proper functional tissue regeneration. Here we engineered biocompatible silicon carbide semiconductive nanowires that synthetically couple, via membrane nanobridge formations, isolated beating cardiomyocytes over distance, restoring physiological cell-cell conductance, thereby permitting the synchronization of bioelectrical activity in otherwise uncoupled cells. Local in-situ multiple injections of nanowires in the left ventricular infarcted regions allow rapid reinstatement of impulse propagation across damaged areas and recover electrogram parameters and conduction velocity. Here we propose this nanomedical intervention as a strategy for reducing ventricular arrhythmia after acute myocardial infarction.

A pathogenic HEXA missense variant in wild boars with Tay-Sachs disease.

Gangliosidoses are inherited lysosomal storage disorders caused by reduced or absent activity of either a lysosomal enzyme involved in ganglioside catabolism, or an activator protein required for the proper activity of a ganglioside hydrolase, which results in the intra-lysosomal accumulation of undegraded metabolites. We hereby describe morphological, ultrastructural, biochemical and genetic features of GM2 gangliosidosis in three captive bred wild boar littermates. The piglets were kept in a partially-free range farm and presented progressive neurological signs, starting at 6 months of age. Animals were euthanized at approximately one year of age due to their poor conditions. Neuropathogens were excluded as a possible cause of the signs. Gross examination showed a reduction of cerebral and cerebellar consistency. Central (CNS) and peripheral (PNS) nervous system neurons were enlarged and foamy, with severe and diffuse cytoplasmic vacuolization. Transmission electron microscopy (TEM) of CNS neurons demonstrated numerous lysosomes, filled by parallel or concentric layers of membranous electron-dense material, defined as membranous cytoplasmic bodies (MCB). Biochemical composition of gangliosides analysis from CNS revealed accumulation of GM2 ganglioside; furthermore, Hex A enzyme activity was less than 1% compared to control animals. These data confirmed the diagnosis of GM2 gangliosidosis. Genetic analysis identified, at a homozygous level, the presence of a missense nucleotide variant c.1495C > T (p Arg499Cys) in the hexosaminidase subunit alpha gene (HEXA), located within the GH20 hexosaminidase superfamily domain of the encoded protein. This specific HEXA variant is known to be pathogenic and associated with Tay-Sachs disease in humans, but has never been identified in other animal species. This is the first report of a HEXA gene associated Tay-Sachs disease in wild boars and provides a comprehensive description of a novel spontaneous animal model for this lysosomal storage disease.

Modeling pulmonary fibrosis through bleomycin delivered by osmotic minipump: a new histomorphometric method of evaluation.

The systemic delivery of bleomycin (BLM) to mice through subcutaneously implanted osmotic minipumps may be used to experimentally mimic the typical features of systemic sclerosis and related interstitial lung diseases. The published studies on this model principally have focused on induced dermal modifications, probably because lung lesions are typically mild, subpleurally localized, and difficult to analyze. The use of high BLM doses to increase their severity has been proposed but is ethically questionable because of the compromising of animal welfare. We propose a tailored histomorphometric method suitable to detect and quantify this type of mild lung lesions. Using a two-step automated image analysis, a peripheral region of interest with a depth of 250 µm from the pleural edge was defined on whole slide images, and the fibrotic foci were histomorphometrically characterized. The effects of different BLM doses on lung alterations were evaluated in C57BL/6 mice and 60 U/kg resulted in a fair compromise between fibrotic lesions and animal welfare. This dose was also tested in time course experiments. The analysis revealed a peak of histological fibrotic-like alterations, cytokine expression, metalloprotease, and macrophagic activation between the 21st and 28th day after pump implant. The induced dermal fibrosis was characterized by the progressive loss of the white dermal adipose layer, an increase in dermal thickness, dermal hyperplasia, and more compacted collagen fibers. Despite the trend toward spontaneous resolution, our model allowed a double organ readout of the BLM effect and the identification of a therapeutic window for testing pharmacological compounds without using life-threatening doses.

Immunolocalization of Orexin A and its receptors in the different structures of the porcine ovary.

Orexins are neuropeptides with pleiotropic functions, involved in the coordination of multiple versatile physiological processes, in particular related to food intake and several aspects of the reproductive process. Their actions are carried out through the bond with the related Orexin 1 (OXR1) and Orexin 2 (OXR2) G-protein-coupled receptors. Studies on the expression of the orexinergic system in the female genital organs are scarce and limited to preovulatory gametogenic follicles and corpora lutea isolated from the rest of the ovary. As the description of only these structures is insufficient to provide a complete picture of the organ, the present study is aimed to give a panoramic view of all the ovarian structures and cells expressing Orexin A (OXA) and its receptors in their original localization. Double labeling immunofluorescent methods, applied on frozen sections of the whole organ in both follicular and luteal phase, were used to highlight the particular distribution and colocalization of the proteins. For a better recognition of cellular morphology and a better distinction between gametogenic (healthy) and atretic follicles, also a single labeling immunolocalization of OXA on formalin fixed paraffin embedded tissues and a TUNEL staining were performed. The results indicate that OXA and its two receptors subtypes are expressed in all the different structures composing the swine ovary, albeit in different ways, in both phases of the ovarian cycle. In general, OXA and OXR2 appear diffusely distributed within "health", proliferating and steroid producing cells, while has granular appearance, being presumably associated to cytoplasmic vesicles, in degenerating cells, independently if apoptotic or not. The immunoreactivity for OXR1, instead, is often associated with the nuclear envelope but it is also detectable, to a lesser extent, diffusely distributed in the cytoplasm of growing or steroid producing cells. When cells undertake the path leading to degeneration, also OXR1 immunoreactivity assumes a granular appearance in the cytoplasm and is colocalized with OXA and OXR2. Different roles for the two receptors in the same cell and a different regulation of their expression remain to be investigated. Their comprehension could help studies of follicle development in pig, as part of in vitro oocyte maturation and fertilization programs in livestock.

Striated Perineal Muscles: Location of Somatic and Autonomic Neurons Projecting to the Male Pig Ischiocavernous Muscle. Neurochemical Features of the Sympathetic Subset.

The location, number and size of the central and peripheral neurons innervating the ischiocavernous muscle (ICM) were studied in male pigs by means of Fast Blue (FB) retrograde neuronal tracing. Moreover the immunohistochemical properties of the sympathetic ganglia were investigated combining the double immunolabeling method. After injection of FB into the left ICM, a mean number of 245.3 ± 134.9 labeled neurons were found in the ipsilateral ventral horn of the S1-S3 segments of the spinal cord (SC), 129.7 ± 45.5 in the L6-S3 ipsilateral and S2-S3 contralateral spinal ganglia (SGs), 2279.3 ± 622.1 in the ipsilateral L2-S2 and contralateral L5-S2 sympathetic trunk ganglia (STGs), 541.7 ± 158 in the bilateral caudal mesenteric ganglia (CMGs), and 78.3 ± 35.8 in the microganglia of the pelvic plexus (PGs). The mean area of the ICM projecting neurons was 1217 ± 69.7 µm2 in the SC, 2737.5 ± 176.5 µm2 in the SGs, 982.8 ± 36.8 µm2 in the STGs, 865.9 ± 39.14 µm2 in the CMGs and 426.2 ± 24.72 µm2 in the PGs. The FB positive neurons of autonomic ganglia contained Dopamine ß hydroxylase, vesicular acetylcholine transporter, neuronal nitric oxyde sinthase, calcitonine gene related peptide, leu-enkephaline, neuropeptide Y, substance P, vasoactive intestinal polypeptide, and somatostatine often colocalized with tyrosine hydroxylase. The particular localization of the motor somatic nucleus, the abundant autonomic innervation and the qualitatively different content of ICM projecting sympathetic neurons suggest a complex regulation of this striated muscle involved in involuntary functions, such as the erection, ejaculation, micturition and defecation. Anat Rec, 301:837-848, 2018. © 2017 Wiley Periodicals, Inc.

Immunophenotypical characterization of canine mesenchymal stem cells from perivisceral and subcutaneous adipose tissue by a species-specific panel of antibodies.

Immunophenotypical characterization of mesenchymal stem cells is fundamental for the design and execution of sound experimental and clinical studies. The scarce availability of species-specific antibodies for canine antigens has hampered the immunophenotypical characterization of canine mesenchymal stem cells (MSC). The aim of this study was to select a panel of species-specific direct antibodies readily useful for canine mesenchymal stem cells characterization. They were isolated from perivisceral and subcutaneous adipose tissue samples collected during regular surgeries from 8 dogs. Single color flow cytometric analysis of mesenchymal stem cells (P3) deriving from subcutaneous and perivisceral adipose tissue with a panel of 7 direct anti-canine antibodies revealed two largely homogenous cell populations with a similar pattern: CD29+, CD44+, CD73+, CD90+, CD34-, CD45- and MHC-II- with no statistically significant differences among them. Antibody reactivity was demonstrated on canine peripheral blood mononuclear cells. The similarities are reinforced by their in vitro cell morphology, trilineage differentiation ability and RT-PCR analysis (CD90+, CD73+, CD105+, CD44+, CD13+, CD29+, Oct-4+ gene and CD31- and CD45- expression). Our results report for the first time a comparison between the immunophenotypic profile of canine MSC deriving from perivisceral and subcutaneous adipose tissue. The substantial equivalence between the two populations has practical implication on clinical applications, giving the opportunity to choose the source depending on the patient needs. The results contribute to routine characterization of MSC populations grown in vitro, a mandatory process for the definition of solid and reproducible laboratory and therapeutic procedures.

Immunohistochemical Properties of the Peripheral Neurons Projecting to the Pig Bulbospongiosus Muscle.

Retrograde neuronal tracing and single labelling immunofluorescence methods were used to define the neurochemical content of the peripheral autonomic and sensitive neurons projecting to the male pig striated bulbospongiosus muscle (BSM). The retrograde fluorescent neuronal tracer Fast Blue (FB) was injected into the left bulbospongiosus muscle of four intact impuberal pigs. After a 10-day survival time, the ipsilateral sacral sympathetic trunk ganglia (STGs), the caudal mesenteric ganglion (CMG), and the sacral spinal ganglia (SGs) were collected from each animal. In FB+ neurons of these ganglia, the presence of cathecolamine- (tyrosine hydroxylase-TH), acetylcholine- (vesicular acetylcholine transporter-VChAT), or nitric oxide-synthesizing (neuronal Nitric Oxide Synthase-nNOS) enzymes and of some biologically active peptides (calcitonine gene-related peptide-CGRP, Leu-Enkephaline-LENK, Neuropeptide Y-NPY, Substance P-SP and Vasoactive Intestinal Peptide-VIP) were studied. The ipsilateral STGs FB+ neurons showed immunoreactivity principally for TH and NPY and in decreasing order for VIP, VChAT, SP, CGRP, nNOS, and LENK. The left CMG FB+ neurons were immunoreactive to TH and NPY, and in smaller proportions for VIP, LENK, VChAT, CGRP, nNOS, and SP. The ipsilateral SGs FB+ neurons resulted immunoractive for CGRP, LENK, NPY, nNOS, SP, and VChAT. The heterogeneous neurochemical content of the peripheral neurons projecting to the pig BSM allows us to hypothesize the involvement of autonomic ganglia in the activity of both blood vessels and striated fibers of the muscle and the involvement of sensory ganglia in the afferent transmission from the muscle to spinal cord and in antidromic mechanisms that causes the relaxation of the BSM blood vessels. Anat Rec, 299:1192-1202, 2016. © 2016 Wiley Periodicals, Inc.

Morphological analysis of the urethral muscle of the male pig with relevance to urinary continence and micturition.

To investigate whether the pig could be considered a suitable model to study lower urinary tract function and dysfunction, the pelvic urethra of 24 slaughtered male pigs were collected, and the associated muscles were macroscopically, histologically and histochemically analyzed. In cross-sections of the urethra, a muscular complex composed of an inner layer of smooth muscle and an outer layer of striated muscle that are not separated by fascial planes was observed. A tunica muscularis, composed of differently oriented smooth muscle bundles, is only evident in the proximal part of the pelvic urethra while, in the remaining part, it contributes to form the prostatic fibromuscular stroma. The striated urethral muscle surrounds the pelvic urethra in a horseshoe-like configuration with a dorsal longitudinal raphe, extending from the bladder neck to the central tendon of perineum. Proximally to the bladder, it is constituted of slow-twitch and fast-twitch myofibers of very small diameter, and embedded in an abundant collagen and elastic fiber net. Moving caudally it is gradually encircled and then completely substituted by larger and compact myofibers, principally presenting circular orientation and fast-twitch histochemical characteristics. So, like in humans, the cranial tract of the muscular system surrounding the pelvic urethra is principally composed of smooth musculature. The striated component cranially may have a role in blocking retrograde ejaculation, while the middle and caudal tracts may facilitate urine and semen flow, and seem especially concerned with the rapid and forceful urethral closure during active continence. Some differences in the morphology and structure between pigs and humans seem due to the different morphology of the 'secondary' sexual organs that develop from the urethral wall and to the different effect of gravity on the mechanics of the urinary system in quadruped and bipedal mammals.

Neurochemical features of the autonomic neurons projecting to the cremaster muscle of the boar.

The cremaster muscle (CM) is a striated muscle showing some unusual features for ordinary striated muscles, in fact it receives, besides somatic innervation, a conspicuous autonomic sympathetic innervation. The autonomic neurons associated with the CM of 4 male intact pigs were typified combining the retrograde nontrans-synaptic fluorescent tracer Fast Blue (FB) and double labeling immunohistochemical methods. We collected the L4 sympathetic trunk ganglion (STG), that our preliminary studies proved to contain the highest number (575.5 ± 152.93; mean ± S.E.M., n = 4) of FB+ sympathetic neurons projecting to CM. About half of the CM projecting neurons of this ganglion were catecholaminergic and showed the colocalization of Tyrosine Hydroxylase (TH) with Neuropeptide Y (NPY), Leu-Enkephaline (LENK), Vasoactive Intestinal Polypeptide (VIP), Calcitonine Gene Related Peptide (CGRP), Substance P (SP), neuronal Nitric Oxyde Sinthase (n-NOS), and Vesicular Acetylcholine Transporter (VAChT). The noncatecholaminergic neurons were immunoreactive for all the other markers tested, even if in small percentages. The conspicuous and heterogeneous contribution of the sympathetic autonomic neurons to the muscle innervation is consistent with the hypothesis of a possible origin of the CM fibers by transdifferentiation of the smooth muscle-like gubernaculum mesenchyma into striated myotubes, suggesting that the cremaster myogenesis is independent from that of the abdominal muscles.

Localization and neurochemical features of the sympathetic trunk ganglia neurons projecting to the urethral muscle. An experimental study in a porcine animal model.

The striated perineal urethral muscle (UM) is involved in the voluntary control of the micturition requiring complex interactions between afferent and efferent (autonomic and somatic) pathways to store and periodically eliminate urine. Our aim was to define the site, cross sectional area and phenotype of sympathetic trunk ganglia (STG) neurons projecting to the porcine UM, combining retrograde neuronal tracer Fast Blue (FB) and double immunohistochemical labelling methods. The research was carried out on 3 male intact pigs, in which we counted a total number of 4992.67 ± 834.35 (mean ± S.E.M., n = 3) FB+ neurons distributed in the bilateral T12-S3 STG. These neurons were significantly larger in lumbar STG than in the sacral ones. Moreover we highlighted the presence of Dopamine ß hydroxylase (DßH), Vesicular Acetylcholine Transporter (VAChT), neuronal Nitric Oxyde Sinthase (n-NOS), Calcitonine Gene Related Peptide (CGRP), Leu-Enkephaline (LENK), Neuropeptide Y (NPY), Substance P (SP), Vasoactive Intestinal Polypeptide (VIP) and Somatostatine (SOM) and their eventual co-existence with Tyrosine Hydroxylase(TH) in both lumbar and sacral FB+ neurons. In particular, lumbar and sacral STG neurons expressed similar percentages of immunoreactivity for TH, SP and CGRP, but showed significantly different levels of immunoreactivity for NPY, VIP, VAChT, LENK, nNOS, DßH and SOM. Taken together, these data indicate a different contribution of lumbar and sacral pathways in the sympathetic transmission to the boar UM.

Double labelling immunohistochemistry on the sympathetic trunk ganglia neurons projecting to the extrinsic penile smooth musculature of the pig: an experimental study on the retractor penis muscle.

Retrograde neuronal tracing and double labelling immunofluorescence methods were used to define the neurochemical content of sympathetic trunk ganglia neurons projecting to the pig retractor penis muscle, which was taken as an experimental model of the male genital smooth musculature. After the injection of Fast Blue into the bulbo-penile portion of the retractor penis muscle, the eventual co-existence of the catecholaminergic marker tyrosine hydroxylase with calcitonine gene related peptide, leu-enkephalin, neuropeptide Y, neuronal nitric oxide synthase, substance P, vasoactive intestinal polypeptide or vesicular acetylcholine transporter was studied in the ipsilateral S1 sympathetic trunk ganglia, which resulted to contain the greatest number of autonomic retractor penis muscle projecting cells. The observation of Fast Blue positive neurons under the fluorescent microscope allowed the identification of different subpopulations of catecholaminergic and non-catecholaminergic retractor penis muscle-projecting neurons. The majority of catecholaminergic cells contained tyrosine hydroxylase alone, while the remaining part showed co-localization of tyrosine hydroxylase with all the other tested markers. These last neurons were immunoreactive, in decreasing percentages, for neuropeptide Y, leu-enkephalin, neuronal nitric oxide synthase, substance P, calcitonine gene related peptide, vasoactive intestinal polypeptide and vesicular acetylcholine transporter. The majority of non-catecholaminergic neurons were immunonegative for all the tested markers. The remaining non-catecholaminergic cells contained, in decreasing percentages, neuropeptide Y, neuronal nitric oxide synthase, leu-enkephalin, vasoactive intestinal polypeptide, vesicular acetylcholine transporter, substance P and calcitonine gene related peptide. Our findings documented the complexity of the neurochemical interactions that regulate both the motor functions of RPM and the blood flow through the muscle.

Sensory and autonomic neurons project both to the smooth retractor penis and to the striated bulbospongiosus muscles. Neurochemical features of the sympathetic subset.

Aim of the present study was to verify, by means of double retrograde neuronal tracers technique, the hypothesis that a subpopulation of sensory and autonomic neurons send collateral axons to both smooth and striated genital muscles. We also wanted to define the neurochemical content of the eventually retrogradelly double labeled (RDL) neurons in the sympathetic trunk ganglia (STG). We used six intact pigs and we injected the tracer Diamidino Yellow (DY) in the smooth left retractor penis muscle (RPM) and the tracer Fast Blue (FB) in the striated left bulbospongiosus muscle (BSM). Rare (2 ± 0.6) RDL neurons were found in the ipsilateral S2 spinal ganglion (SG), 220 ± 42 in the ipsilateral STGs, from L3 to S3, 19 ± 15 in the contralateral S1-S2 ones and 22 ± 5 in the bilateral caudal mesenteric ganglia (CMG). The RDL neurons of the STG were IR for TH (85 ± 13%), DßH (69 ± 17%), NPY (69 ± 23%), nNOS (60 ± 11%), LENK (54 ± 19%), VIP (53±26%), SOM (40 ± 8%), CGRP (34 ± 12%), SP (31 ± 16%), and VAChT (28 ± 3%). Our research highlights the presence of sensory and sympathetic neurons with qualitatively different neurochemical content sending axons both to the smooth RPM and to the striated BSM of the pig. These RDL neurons are likely to project to the smooth vasal musculature to create the ideal physiological conditions in which these muscles can optimize the erectile function.

Immunohistochemical characteristics of the nerve fibres of sow retractor clitoridis muscle.

The occurrence of several biologically active neuropeptides (calcitonine gene-related peptide, leu-enkephaline, neuropeptide Y, substance P, and vasoactive intestinal peptide) or nitric oxide-synthesizing enzymes (neuronal nitric oxide synthase), tyrosine hydroxylase, vesicular acetylcholine transporter, and their co-localization with tyrosine hydroxylase were investigated by immunohistochemistry in the retractor clitoridis muscle of slaughtered sows. Single immunolabelling revealed that tyrosine hydroxylase and neuropeptide Y immunoreactive nerve fibres were the most numerous, followed by the neuronal nitric oxide synthase and calcitonine gene-related peptide immunoreactive ones, the vasoactive intestinal peptide, substance P and leu-enkephaline immunoreactive nerve fibres were few and vesicular acetylcholine transporter immunoreactivity were observed only in single fibres. Double immunolabelling revealed the only co-localization of tyrosyne hydroxylase with neuropeptide Y. The most reliable labelling of nerve fibres of the retractor clitoridis muscle was observed around blood vessels, followed by non-vascular smooth muscles. The present data indicate that the sow retractor clitoridis muscle receives nerve fibres that exhibit different chemical codes and, likely, differences in their chemical coding depend on the target-structure.

Localization of the autonomic, somatic and sensory neurons innervating the cranial tibial muscle of the pig.

The location of sympathetic, somatic and sensory neurons projecting to the cranial tibial muscle of the pig hindlimb was studied with the neuronal non-transynaptic tracer Fast Blue. Additionally, the number and the size of these neurons were determinated. The Fast blue, randomly applied to the cranial tibial muscle belly of 3 pigs, labelled sympathetic neurons in the ipsilateral L5-S3 and contralateral S1 sympathetic trunk ganglia and in the prevertebral caudal mesenteric ganglia of both sides. The somatic motoneurons were identified in the ipsilateral ventral horn of the S1 segment of spinal cord, while the sensory neurons were located in the ipsilateral L7-S1 spinal ganglia. The diameter of the multipolar sympathetic neurons oscillated between 26 and 46 microm in the sympathetic trunk ganglia and between 18 and 42 microm in the caudal mesenteric ganglia. The size of the multipolar spinal motoneurons oscillated between 33 and 102 microm. The size of the pseudounipolar sensory neurons oscillated between 23 and 67 microm. In all ganglia, the labelled neurons were localized at random and did not show a somatotopic distribution. Our results document a conspicuous autonomic innervation projecting to the "classic" skeletal cranial tibial muscle. Probably this innervation is destined to the muscle vessels.

Striated perineal muscles: location of autonomic, sensory, and somatic neurons projecting to the male pig bulbospongiosus muscle.

The location, number, and size of the neurons innervating the bulbospongiosus muscle (BSM) were studied in male pigs, by means of Fast Blue (FB) retrograde transport. After injection of FB into the left BSM, labeled neurons were found bilaterally in the L2-S4 sympathetic trunk ganglia (STGs), in the caudal mesenteric ganglia (CMGs), in the microganglia of the pelvic plexus (PGs), in a dorsolateral area with respect to the central canal of S1-S3 segments of the spinal cord (SC) and in the S1-S4 ipsilateral and S2-S3 contralateral spinal ganglia (SGs). The mean number of labeled FB cells was 3,122 +/- 1,968 in STGs, 979 +/- 667 in CMGs, 108 +/- 104 in PGs, 89 +/- 39 in SC and 77 +/- 23 in SGs. The area of the multipolar neurons was 852 +/- 22 microm(2) in the STGs, 878 +/- 23 microm(2) in the CMGs and 922 +/- 31 microm(2) in the PGs. The multipolar SC neurons had an area of 1,057 +/- 38 microm(2), while pseudounipolar SG cells had dimensions of 2,281 +/- 129 microm(2). Our research enables us to highlight two peculiarities regarding the innervation of the boar BSM: the very high number of labeled autonomic neurons and the particular localization of the motor somatic nucleus.

Experimental study to localize the neurons projecting to the lamb retractor penis muscle.

Aim of the present study was to locate the neurons projecting to the lamb retractor penis muscle, a smooth muscle associated to the penis. The retrograde neuronal tracer Fast Blue was injected into the bulbopenile portion of the left retractor penis muscle. Labelled cells were found bilaterally in the S2-S4 spinal ganglia, from the last two lumbar (L5-L6 or L6-L7) to S4 sympathetic trunk ganglia and in the hypogastric and pelvic plexuses. Fast blue-positive (FB+) neurons were also found in the intermediate gray substance in the S1-S4 segments of the spinal cord. Our research enables us to describe the organization of the innervation of the lamb retractor penis muscle, highlighting the site of the primary afferent, postganglionic efferent and presumably preganglionic parasympathetic neurons projecting to the muscle.

Sensory, motor somatic, and autonomic neurons projecting to the porcine cremaster muscle.

The location of sensory, somatic, and autonomic neurons projecting to the pig cremaster muscle (CM) was studied by means of the retrograde neuronal tracer Fast Blue (FB) technique. FB was randomly injected in the left CM of four impuberal pigs and serial sections of sensory and autonomic ganglia and spinal cord were examined under a fluorescence microscope. Additionally, some indications about the number and size of labeled neurons were given. Sensory pseudounipolar somata were located ipsilaterally in the L2-L6 and S1-S2 dorsal root ganglia, their total number ranging between 125 and 194, their mean diameter between 24 and 89 microm. Somatic multipolar motoneurons were located ipsilaterally in the L2-L4 neuromeres of the spinal cord, their total number ranging between 53 and 169, their mean diameter between 29 and 53 microm. Autonomic multipolar paravertebral ganglia neurons were located ipsilaterally from L1 to S4 and contralaterally from L2 to S2. Their total number ranged from 2,015 to 3,067 and their mean diameter between 25 and 55 microm. The multipolar caudal mesenteric ganglia neurons were located bilaterally, their total number ranging between 14 and 1,408 and their diameter from 22 to 39 microm. In two subjects only, multipolar neurons were also found ipsilaterally in the microganglia of pelvic plexus (2 and 13 neurons). Their mean diameter ranged between 28 and 54 microm. Our study documented that the CM-projecting neurons were located at different neural levels, with a predominance in the autonomic ganglia.

Peripheral neurons innervating the extrinsic smooth penile musculature of the pig: experimental study by retrograde transport and immunohistochemistry.

Peripheral autonomic and sensitive neurons projecting to the extrinsic smooth penile musculature of the pig were studied by means of retrograde tracing and single-labelling immunofluorescence methods. The fluorescent retrograde tracer Fast Blue was injected into the left retractor penis muscle, that was taken as an experimental model of the male genital smooth musculature, of 4 castrated pigs. After a 7 day survival time, the ipsilateral paravertebral ganglion S1, the caudal mesenteric ganglion and the dorsal root ganglion S2 were collected. In these ganglia, the presence and the distribution of immunoreactivities to cathecolamine- (Tyrosine Hydroxylase), acetylcholine- (Vesicular Acetylcholine Transporter), or nitric oxide-synthesizing (neuronal Nitric Oxide Synthase) enzymes and to some biologically active peptides (Calcitonine Gene-Related Peptide, Leu-Enkephaline, Neuropeptide Y, Substance P and Vasoactive Intestinal Peptide) were studied. In paravertebral ganglion S1, Tyrosine Hydroxylase and Neuropeptide Y were the most frequently present substances. Also Leu-Enkephaline and neuronal Nitric Oxide were present quite frequently, while there was scarce immunoreactivity for the other antisera (in decreasing order Substance P, Vasoactive Intestinal Peptide, Vesicular Acetylcholine Transporter, Calcitonine Gene-Related Peptide). In caudal mesenteric ganglion, in addition to Tyrosine Hydroxylase- and Neuropeptide Y-immunoreactivity, Substance P-, Vesicular Acetylcholine Transporter-, Vasoactive Intestinal Peptide-, Leu-Enkephaline- immunoreactivity were also frequently present, followed by neuronal Nitric Oxide- and Calcitonine Gene-Related Peptide- immunoreactivity. In dorsal root ganglion S2, Calcitonine Gene-Related Peptide and neuronal Nitric Oxide resulted to be the most frequently present neurotransmitters, followed by Vasoactive Intestinal Peptide, Vesicular Acetylcholine Transporter, Leu-Enkephaline, Substance P, Tyrosine Hydroxylase and Neuropeptide Y.