Xanthohumol, a prenylated flavonoid from hops (Humulus lupulus L.) exerts multidirectional pro-healing properties towards damaged zebrafish hair cells by regulating the innate immune response.

Xanthohumol (XN) is a prominent prenylated flavonoid present in the hop plant (Humulus lupulus L.). Despite undoubted pro-healing properties of hop plant, there is still a need for clinical investigations confirming these effects as well as the underlying molecular mechanisms. The present study was designed to (1) establish the role of XN in non-invasive inflammation induced by chemical damage to zebrafish hair cells, (2) clarify if it influences cell injury severity, neutrophil migration, macrophage activation, cell regeneration, and (3) find out whether it modulates the gene expression profile of chosen immune and stress response markers. All experiments were performed on 3 dpf zebrafish larvae. After fertilization the embryos were transferred to appropriate XN solutions (0.1 µM, 0.3 µM and 0.5 µM). The 40 min 10 µM CuSO4 exposure evoked severe damage to posterior lateral line hair cells triggering a robust acute inflammatory response. Four readouts were selected as the indicators of XN role in the process of inflammation: 1) hair cell death, 2) neutrophil migration towards damaged hair cells, 3) macrophage activation and recruitment to damaged hair cells, 4) hair cell regeneration. The assessments involved in vivo confocal microscopy imaging and qPCR based molecular analysis. It was demonstrated that XN (1) influences death pathway of damaged hair cells by redirecting their severe necrotic phenotype into apoptotic one, (2) impacts the immune response via regulating neutrophil migration, macrophage recruitment and activation (3) modulates gene expression of immune system markers and (4) accelerates hair cell regeneration.

The Effect of Simvastatin on the Dynamics of NF-κB-Regulated Neurodegenerative and Neuroprotective Processes in the Acute Phase of Ischemic Stroke.

Statins are lipid-lowering drugs that act by inhibiting 3-hydroxy-3-methylglutaryl coenzyme A reductase, a rate-limiting enzyme in cholesterol biosynthesis. Animal studies have shown neuroprotective effects of statins in cerebral stroke. However, the underlying mechanisms are not fully understood. The nuclear factor-kappa B (NF-κB) transcription factor is involved in the regulation of apoptosis in stroke. Different dimers of NF-κB regulate the gene expression of proteins involved in both neurodegeneration and neuroprotection. We aimed to determine whether simvastatin improves stroke outcome via inhibition of the RelA/p65-containing subunit and downregulation of stroke-induced pro-apoptotic genes or via activation of NF-κB dimers containing the c-Rel subunit and upregulation of anti-apoptotic genes during the acute stroke phase. Eighteen-month-old Wistar rats, subjected to permanent MCAO or sham surgery, were administered simvastatin (20 mg/kg b.w.) or saline for 5 days before the procedure. Stroke outcome was determined by measuring cerebral infarct and assessing motor functions. The expression of NF-κB subunits in various cell populations was investigated using immunofluorescence/confocal microscopy. RelA and c-Rel were detected by WB. The NF-κB-DNA binding activity was investigated using EMSA, and expression of Noxa, Puma, Bcl-2, and Bcl-x genes was analyzed by qRT-PCR. Results showed a 50% infarct size reduction and significant motor function improvement in the simvastatin-treated animals which correlated with a decrease in RelA and a transient increase in the c-Rel level in the nucleus, normalization of the NF-κB-DNA binding activity, and downregulation of the NF-κB-regulated genes. Our results provide new insights into the statin-mediated neuroprotective action against stroke based on NF-κB pathway inhibition.

Kisspeptin and GPR54 Receptor Expression in Endometrial Cancer Tissue.

Kisspeptin (KISS) is a natural peptide-discovered in 1996 as a factor inhibiting the ability to metastasize in malignant melanoma. This protein plays also a regulatory role in the process of puberty, the menstrual cycle, spermatogenesis, implantation and development of the human placenta. The present study aimed to evaluate the expression of KISS and its receptor GPR54 in endometrial cancer (EC) tissue, depending on the histological type of cancer, its stage, various demographic characteristics, and clinical conditions in 214 hysterectomy patients. Expression of KISS and GPR54 was confirmed in 99.5% and 100% of the cases, respectively. Hormone replacement therapy and the coexistence of the anti-Müllerian type 2 receptor in cancer tissue enhanced KISS expression. Smoking, on the other hand, decreased KISS expression. GPR54 expression increased with the advancement of the disease (according to FIGO classification). Also, the presence of the anti-Müllerian type 2 receptor in EC increased the level of GPR54. Hypertension, age and miscarriage harmed the presence of GPR54. The histological type of cancer, diabetes type 2, body mass index, hormonal contraception, number of deliveries, birth weight of newborns, breastfeeding time, and the presence of AMH in EC tissue were not associated with the expression of either KISS nor GPR54. The KISS level was also significantly related to the GPR54 level. Considering that KISS is a non-toxic peptide with antimetastatic properties, further investigation is essential to determine the clinical significance of this peptide.

Morphology and immunohistochemical characteristics of the otic ganglion in the chinchilla (Chinchilla laniger Molina).

INTRODUCTION: The available literature provides relatively little information on the morphology of the autonomic head ganglia in rodents including their neurochemical codding. MATERIAL AND METHODS: Morphological investigations of the otic ganglion of the chinchilla were performed using the modified acetylcholinesterase method. The cellular structure was investigated with histological techniques and neurochemical properties were studied with the double-labelling immunofluorescence method. RESULTS: Macromorphological investigations allowed the otic ganglion to be identified as a compact, oval agglomeration of neurons and nerve fibers. Multidimensional cross-sections revealed densely arranged neuronal perikarya and two populations of nerve cells differing in size were distinguished. The large cells (40-50 µm) accounted for about 80% of the neurons in the cross-sections. Moreover, a small number of intraganglionic nerve fibers was observed. Immunohistochemical staining revealed that over 85% of the neuronal cell bodies in the otic ganglion contained immunoreactivity to VAChT or ChAT. VIP-immunoreactive perikarya comprised approximately 10% of the ganglionic cells. Double staining revealed the presence of VAChT+ and NOS+ neurons which amounted to about 45% of the nerve cells in the otic ganglion. NOS+ only perikarya comprised approx. 15% of all the neurons. Immunoreactivity to enkephalins, substance P, somatostatin, and galanin was expressed in single nerve cell bodies and nerve fibers except numerous substance P+ intraganglionic nerve fibers. Some of them were stained also for CGRP. Single neurons stained for tyroxine hydroxylase. CONCLUSIONS: Our results, compared with findings in other rodent species suggest the existence of interspecies differences in the morphology, cellular structure, and immunohistochemical properties of the head autonomic ganglia in mammals.

The Effect of Castration on Peripheral Autonomic Neurons Supplying Mammalian Male Genitourinary System.

This review paper deals with the influence of androgens (testosterone) on pelvic autonomic pathways in male mammals. The vast majority of the relevant information has been gained in experiments involving castration (testosterone deprivation) performed in male rats, and recently, in male pigs. In both species, testosterone significantly affects the biology of the pathway components, including the pelvic neurons. However, there are great differences between rats and pigs in this respect. The most significant alteration is that testosterone deprivation accomplished a few days after birth results some months later in the excessive loss (approximately 90%) of pelvic and urinary bladder trigone intramural neurons in the male pig, while no changes in the number of pelvic neurons are observed in male rats (rats do not have the intramural ganglia). In the castrated pigs, much greater numbers of pelvic neurons than in the non-castrated animals express CGRP, GAL, VIP (peptides known to have neuroprotective properties), and caspase 3, suggesting that neurons die due to apoptosis triggered by androgen deprivation. In contrast, only some morpho-electrophysiological changes affecting neurons following castration are found in male rats. Certain clinicopathological consequences of testosterone deprivation for the functioning of urogenital organs are also discussed.

The influence of castration on intramural neurons of the urinary bladder trigone in male pigs.

The present study investigated the influence of castration performed at neonatal age on neuronal elements in the intramural ganglia of the urinary bladder trigone (UBT) in male pigs using double-labeling immunohistochemistry. The ganglia were examined in intact (IP) 7-day-old (castration day) pigs, and at 3 and 6 months after surgery. In IP and control (3- and 6-month-old noncastrated pigs) groups, virtually, all neurons were adrenergic (68%) or cholinergic (32%) in nature. Many of them (32%, 51%, and 81%, respectively; 56%, 75%, and 85% adrenergic; and 32%, 52%, and 65% cholinergic, respectively) stained for the androgen receptor (AR), and only a small number of nerve cells were caspase-3 (CASP-3)-positive. In 3- and 6-month-old castrated pigs, an excessive loss (87.6% and 87.5%, respectively) of neurons and intraganglionic nerve fibers was observed. The majority of the surviving adrenergic (61% and 72%, respectively) and many cholinergic (41% and 31%, respectively) neurons expressed CASP-3 and were also AR-positive (61% and 66%, and 40% and 36%, respectively). This study revealed for the first time the excessive loss of intramural UBT neurons following castration, which could have resulted from apoptosis induced by androgen deprivation.

P-Glycoprotein Inhibitor Tariquidar Plays an Important Regulatory Role in Pigmentation in Larval Zebrafish.

Zebrafish has emerged as a powerful model in studies dealing with pigment development and pathobiology of pigment diseases. Due to its conserved pigment pattern with established genetic background, the zebrafish is used for screening of active compounds influencing melanophore, iridophore, and xanthophore development and differentiation. In our study, zebrafish embryos and larvae were used to investigate the influence of third-generation noncompetitive P-glycoprotein inhibitor, tariquidar (TQR), on pigmentation, including phenotype effects and changes in gene expression of chosen chromatophore differentiation markers. Five-day exposure to increasing TQR concentrations (1 µM, 10 µM, and 50 µM) resulted in a dose-dependent augmentation of the area covered with melanophores but a reduction in the area covered by iridophores. The observations were performed in three distinct regions-the eye, dorsal head, and tail. Moreover, TQR enhanced melanophore renewal after depigmentation caused by 0.2 mM 1-phenyl-2-thiourea (PTU) treatment. qPCR analysis performed in 56-h post-fertilization (hpf) embryos demonstrated differential expression patterns of genes related to pigment development and differentiation. The most substantial findings include those indicating that TQR had no significant influence on leukocyte tyrosine kinase, GTP cyclohydrolase 2, tyrosinase-related protein 1, and forkhead box D3, however, markedly upregulated tyrosinase, dopachrome tautomerase and melanocyte inducing transcription factor, and downregulated purine nucleoside phosphorylase 4a. The present study suggests that TQR is an agent with multidirectional properties toward pigment cell formation and distribution in the zebrafish larvae and therefore points to the involvement of P-glycoprotein in this process.

Inferior vagal ganglion galaninergic response to gastric ulcers.

Galanin is a neuropeptide widely expressed in central and peripheral nerves and is known to be engaged in neuronal responses to pathological changes. Stomach ulcerations are one of the most common gastrointestinal disorders. Impaired stomach function in peptic ulcer disease suggests changes in autonomic nerve reflexes controlled by the inferior vagal ganglion, resulting in stomach dysfunction. In this paper, changes in the galaninergic response of inferior vagal neurons to gastric ulceration in a pig model of the disease were analyzed based on the authors' previous studies. The study was performed on 24 animals (12 control and 12 experimental). Gastric ulcers were induced by submucosal injections of 40% acetic acid solution into stomach submucosa and bilateral inferior vagal ganglia were collected one week afterwards. The number of galanin-immunoreactive perikarya in each ganglion was counted to determine fold-changes between both groups of animals and Q-PCR was applied to verify the changes in relative expression level of mRNA encoding both galanin and its receptor subtypes: GalR1, GalR2, GalR3. The results revealed a 2.72-fold increase in the number of galanin-immunoreactive perikarya compared with the controls. Q-PCR revealed that all studied genes were expressed in examined ganglia in both groups of animals. Statistical analysis revealed a 4.63-fold increase in galanin and a 1.45-fold increase in GalR3 mRNA as compared with the controls. No differences were observed between the groups for GalR1 or GalR2. The current study confirmed changes in the galaninergic inferior vagal ganglion response to stomach ulcerations and demonstrated, for the first time, the expression of mRNA encoding all galanin receptor subtypes in the porcine inferior vagal ganglia.

Nerve structures of the heart and their immunohistochemical characterization in 10-week-old porcine foetuses.

The aim of the present study was to investigate the distribution of neuronal perikarya and nerve fibres and their chemical coding in the heart of 10-week-old porcine foetuses. The foetuses, obtained from a local slaughterhouse, were fixed by immersion in 4% buffered (pH 7.4) paraformaldehyde. Cryostat sections of the hearts were processed for single- or double-labelling immunofluorescence methods using antibodies against protein gene product (PGP) dopamine beta-hydroxylase (DßH), acetylcholine vesicular transporter (VAChT) and calcitonin gene-related peptide (CGRP). Numerous clusters of the PGP-positive nerve cells were observed throughout the heart wall. The majority of the clusters were found beneath the epicardium around the root of the aorta, pulmonary trunk and main veins. Some single PGP-positive neurons or small clusters of nerve cells were observed in the epicardium of heart atria and ventricles. The richest network of PGP-positive nerve fibres was observed in the base of the heart near the main cardiac blood vessel openings. Many bundles of PGP-positive nerve fibres were observed throughout the four cardiac chambers. Double-immunohistochemical staining revealed that the majority of the neurons contained immunoreactivity to VAChT, some of them stained for CGRP and the single neuronal somata were DßH-positive. The nerve fibres supplying the heart expressed immunoreactivity to DßH, VAChT or CGRP. The distribution and neurochemical coding of the nerve structures in ten-week-old foetuses are different from those observed in the hearts of juvenile pigs.

L'objectif du présent étude était d'examiner la disposition des cellules et des fibres nerveuses, ainsi que de leur codage, aux cœurs de fœtus de porc de 10 semaines. Les fœtus, obtenus dans un abattoir locale, ont été fixés par immersion dans la solution de paraformaldéhyde 4% (pH 7,4). Des coupes de cœurs, obtenues à l'aide du cryostat, ont subi une coloration immunohistochimique simple ou double afin de dépister, dans des structures nerveuses, la présence du produit génétique protéiné (PGP), de la dopamine bêta-hydroxylase (DßH), du transporteur vésiculaire de l'acétylcholine (VAChT) et du peptide relié au gène calcitonine (CGRP). De nombreuses concentrations de cellules nerveuses PGP-positives ont été observées dans la paroi cardiaque. La plupart des concentrations ont été retrouvées en dessous de l'épicarde, autour de la racine de l'aorte, du tronc pulmonaire et des veines principales. Des neurones singulaires PGP-positifs ou des petites concentrations de cellules nerveuses ont été observées dans l'épicarde des oreillettes et des ventricules du cœur. Le plus riche réseau de fibres nerveuses PGP-positives a été observé dans la base du cœur, près de l'acheminement des vaisseaux principaux. Dans les quatre chambres du cœur, de nombreux faisceaux de fibres nerveuses PGP-positives ont été observées. Des colorations immunohistochimiques doubles ont démontré que la plupart de neurones présentaient une immunoréactivité par rapport à VAChT, certains par rapport à CGRP, et des corps singulaires de cellules nerveuses étaient positives par rapport à DßH. Des fibres nerveuses alimentant le cœur présentaient une immunoréactivité par rapport à DßH, VAChT ou CGRP. La distribution et le codage neurochimique des structures nerveuses chez des fœtus de 10 semaines diffèrent de ceux observés dans les cœurs de jeunes porcs.

Distribution and chemical coding of phoenixin-immunoreactive nerve structures in the spinal cord of the pig.

Phoenixin (PNX) is a newly described peptide found in both neural and non-neural tissues. Until now, no attempts have been made to investigate the expression of PNX in the nervous system of animals other than laboratory rodents, in which an enzyme immunoassay revealed the highest quantity of the substance in the spinal cord. Since the domestic pig, due to its anatomical and histological resemblance to humans, is often used as an animal model in biomedical investigations, the present study was designed to examine PNX-immunoreactivity in the spinal cords of female pigs (n=5). The spinal cords were dissected and divided into the cervical, thoracic, lumbar, sacral and coccygeal segments, which were sectioned transversally into 10-µm-thick serial sections. The sections from each spinal cord segment were processed for double-labelling immunohistochemistry using antibodies against PNX in a mixture with those against calcitonin gene-related peptide (CGRP), substance P (SP) or choline acetyltransferase (CHAT). The PNX-immunoreactivity had a similar distribution in the grey matter of all the spinal cord sections examined and was mainly observed in varicose nerve fibres (NF) that formed a dense plexus in laminae I and II of the dorsal horn. Nearly all of the PNX-immunoreactive NF stained also for CGRP or SP and, interestingly, many of them were CHAT-positive. The present study has provided for the first time the detailed information on the arrangement and chemical features of nerve structures expressing PNX-immunoreactivity in the spinal cord of a large mammal. The exact function of PNX in the spinal cord is not known yet. However, the distribution pattern and immunohistochemical characteristics of PNX-IR NF clearly suggest that this peptite most likely plays a role in spinal noxious signalling.

Effect of castration on pelvic neurons in the male pig.

The present study investigated the influence of castration performed at neonatal age on neuronal elements in the anterior pelvic ganglion of the male pig with immunohistochemistry and quantitative real-time PCR (qPCR). The ganglia were examined 3 and 6 months after surgery. In 3-month-old castrated pigs (3MCP) 74% of adrenergic and 31% of cholinergic neurons stained for caspase-3 (CASP-3), and much greater numbers of perikarya than in the control animals expressed CGRP, galanin (GAL) and VIP (peptides known to have neuroprotective properties). In 6-months-old castrated pigs (6MCP), an excessive loss (90%) of neurons and intraganglionic nerve fibres was found. The survived adrenergic and cholinergic neurons also expressed CASP-3, CGRP, GAL or VIP. The qPCR results corresponded with immunofluorescence findings. In 3MCP, genes for CASP-3 and CGRP were up-regulated, while the expression of those for DßH, VAChT, GAL, VIP and SP displayed statistically insignificant variations. In 6MCP, distinctly up-regulated were genes for CGRP, GAL, VIP, SP, DßH and VAChT, while the expression of casp3 gene was down-regulated. The study revealed for the first time the excessive loss of pelvic neurons following castration, and a realistic assumption is proposed, that the neurons died due to apoptosis triggered by androgen deprivation.

Innervation of internal female genital organs in the pig during prenatal development.

This study investigated the innervation of internal genital organs in 5-, 7- and 10-week-old female pig foetuses using single and double-labelling immunofluorescence methods. The structure and topography of the organs was examined using a scanning electron microscope (SEM). The investigations revealed differences in the innervation between the three developmental periods. Immunostaining for protein gene product 9.5 (PGP; general neural marker) disclosed solitary nerve fibres in the external part of the gonadal ridge and just outside of the mesenchyme surrounding mesonephric ducts in 5-week-old foetuses. Double-labelling immunohistochemistry revealed that nerve fibres associated with the ridge expressed dopamine ß-hydroxylase (DßH; adrenergic marker) or vesicular acetylcholine transporter (VAChT; cholinergic marker). In 7-week-old foetuses, the PGP-positive nerve terminals were absent from the gonad but some of them ran outside and along, and sometimes penetrated into the mesenchyme surrounding the tubal and uterine segments of the paramesonephric ducts and uterovaginal canal. Few axons penetrated into the mesenchyme. DßH-positive fibres were found in single nerve strands or bundles distributed at the edge of the mesenchyme. VAChT-positive nerve terminals formed delicate bundles located at the edge of the mesenchyme, and the single nerves penetrated into the mesenchyme. DßH was also expressed by neurons which formed cell clusters comprising also DßH- or VAChT-positive nerve fibres. In 10-week-old foetuses, PGP-positive nerve fibres were still absent from the ovary but some were distributed in the mesenchyme associated with the uterovaginal canal and uterine and a tubal segment of the paramesonephric ducts, respectively. DßH- or VAChT-positive nerve fibres were distributed at the periphery of the mesenchyme associated with the uterovaginal canal. Some DßH- and many VAChT-positive nerve fibres were evenly distributed throughout the mesenchyme. The clusters of nerve cells comprised DßH-positive perikarya and DßH- or VAChT-positive nerve fibres. The investigations revealed no DßH/VAChT-positive nerve fibres or neurons as well as no nerve structures stained for calcitonin gene-related peptide and/or substance P (sensory markers) associated with the genital organs in the studied prenatal periods.

Pituitary adenylate cyclase-activating polypeptide (PACAP-38) plays an inhibitory role against inflammation induced by chemical damage to zebrafish hair cells.

Pituitary adenylate cyclase-activating polypeptide (PACAP-38) is a common neuropeptide exerting a wide spectrum of functions in many fields, including immunology. In the present study, 5-day post-fertilization (dpf) zebrafish larvae of three diverse genetic lines [transgenic lines Tg(MPX:GFP) with GFP-labelled neutrophils and Tg(pou4f3:GAP-GFP) with GFP-labelled hair cells and the wild-type Tuebingen] were used to investigate an inhibitory role of PACAP-38 in inflammation associated with damaged hair cells of the lateral line. Individuals of each genetic line were assigned to four groups: (1) control, and those consisting of larvae exposed to (2) 10 µM CuSO4, (3) 10 µM CuSO4+100 nM PACAP-38 and (4) 100 nM PACAP-38, respectively. Forty-minute exposure to CuSO4 solution was applied to evoke necrosis of hair cells and consequent inflammation. The inhibitory role of PACAP-38 was investigated in vivo under a confocal microscope by counting neutrophils migrating towards damaged hair cells in Tg(MPX:GFP) larvae. In CuSO4-treated individuals, the number of neutrophils associated with hair cells was dramatically increased, while PACAP-38 co-treatment resulted in its over 2-fold decrease. However, co-treatment with PACAP-38 did not prevent hair cells from extensive necrosis, which was found in Tg(pou4f3:GAP-GFP) individuals. Real-Time PCR analysis performed in wild-type larvae demonstrated differential expression pattern of stress and inflammation inducible markers. The most significant findings showed that CuSO4 exposure up-regulated the expression of IL-8, IL-1ß, IL-6 and ATF3, while after PACAP-38 co-treatment expression levels of these genes were significantly decreased. The presence of transcripts for all PACAP receptors in neutrophils was also revealed. Adcyap1r1a and vipr1b appeared to be predominant forms. The present results suggest that PACAP-38 should be considered as a factor playing an important regulatory role in inflammatory response associated with pathological processes affecting zebrafish hair cells and it cannot be excluded that this interesting property has more universal significance.

Neuropeptides and lymphocyte populations in the porcine ileum and ileocecal lymph nodes during postnatal life.

The maturation-related changes in the concentrations of galanin (Gal), vasoactive intestinal polypeptide (VIP), substance P (SP) and somatostatin (Som), as well as in subpopulations of lymphocytes expressing antigens CD2 (lymphocytes T), CD4 (T helper), CD8 (T cytotoxic), CD21 (B lymphocytes), CD5-/CD8+ (NK cells) and TCRgamma/delta (gut mucosal/intraepitelial cells) were studied in the ileal Peyer's patches and ileo-cecal lymph nodes in female pigs aged 3 days, 2 weeks, 4 weeks and 4 months. As regards neuropeptide concentrations statistically significant changes in the ileum and lymph nodes were found only in case of Gal and VIP. The concentrations of neuropeptides were significantly higher only in new-born animals. As regards the changes in subpopulations of lymphocytes, statistically significant changes were noticed only in 4-months old animals and were dealing only with CD2+ and TCRgamma/delta cells in the ileum as well as CD4+, CD8+, CD21+ and TCRgamma/delta in lymph nodes. The highest number of CD8+, CD21+ and TCRgamma/delta lymphocytes occurred in 4-months old animals.

Efficacy of lateral- versus medial-approach hip joint capsule denervation as surgical treatments of the hip joint pain; a neuronal tract tracing study in the sheep.

OBJECTIVE: To evaluate efficacy of denervation of the of the hip joint capsule (HJC), as a treatment of hip joint pain. Specifically, we tested the hypothesis that HJC denervation will significantly reduce the number of sensory neurons innervating the capsule. STUDY DESIGN: Denervation of the HJC from a medial or lateral approach was followed by retrograde tracing of sensory neurons innervating the capsule. ANIMALS: Twenty adult male sheep (30-40 kg of body weight; Polish merino breed) were used in the study. METHODS: The hip joint was denervated from medial (n = 5) or lateral (n = 5) surgical approaches. Immediately after denervation, the retrograde neural tract tracer Fast Blue (FB) was injected into the HJC. An additional ten animals (n = 5 for medial and n = 5 for lateral approach) received the same treatment without HJC denervation to provide the appropriate controls. RESULTS: Results of the study revealed that the vast majority of retrogradely labelled sensory neurons innervating the HJC originate from fifth lumbar to second sacral dorsal root ganglia. Both the medial and the lateral denervations significantly reduced the number of sensory neurons innervating the HJC (39.2% and 69.0% reduction respectively). CONCLUSIONS: These results show that denervation of the HJC is an effective surgical procedure for reduction of the sensory neuronal input to the HJC. Moreover, the lateral approach was found to be significantly more effective for reducing sensory innervation as compared to the medial one.

The Influence of Resiniferatoxin (RTX) and Tetrodotoxin (TTX) on the Distribution, Relative Frequency, and Chemical Coding of Noradrenergic and Cholinergic Nerve Fibers Supplying the Porcine Urinary Bladder Wall.

The present study investigated the influence of intravesically instilled resiniferatoxin (RTX) or tetrodotoxin (TTX) on the distribution, number, and chemical coding of noradrenergic and cholinergic nerve fibers (NF) supplying the urinary bladder in female pigs. Samples from the bladder wall were processed for double-labelling immunofluorescence with antibodies against cholinergic and noradrenergic markers and some other neurotransmitter substances. Both RTX and TTX caused a significant decrease in the number of cholinergic NF in the urinary bladder wall (in the muscle coat, submucosa, and beneath the urothelium). RTX instillation resulted in a decrease in the number of noradrenergic NF in the submucosa and urothelium, while TTX treatment caused a significant increase in the number of these axons in all the layers. The most remarkable changes in the chemical coding of the NF comprised a distinct decrease in the number of the cholinergic NF immunoreactive to CGRP (calcitonin gene-related peptide), nNOS (neuronal nitric oxide synthase), SOM (somatostatin) or VIP (vasoactive intestinal polypeptide), and an increase in the number of noradrenergic NF immunopositive to GAL (galanin) or nNOS, both after RTX or TTX instillation. The present study is the first to suggest that both RTX and TTX can modify the number of noradrenergic and cholinergic NF supplying the porcine urinary bladder.

The Influence of Tetrodotoxin (TTX) on the Distribution and Chemical Coding of Caudal Mesenteric Ganglion (CaMG) Neurons Supplying the Porcine Urinary Bladder.

The treatment of micturition disorders creates a serious problem for urologists. Recently, new therapeutic agents, such as neurotoxins, are being considered for the therapy of urological patients. The present study investigated the chemical coding of caudal mesenteric ganglion (CaMG) neurons supplying the porcine urinary bladder after intravesical instillation of tetrodotoxin (TTX). The CaMG neurons were visualized with retrograde tracer Fast blue (FB) and their chemical profile was disclosed with double-labeling 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 TTX-treated pigs (n = 6), the vast majority (92.6% ± 3.4% and 88.8% ± 2%, respectively) of FB-positive (FB+) nerve cells were TH+. TTX instillation caused a decrease in the number of FB+/TH+ neurons immunopositive to NPY (88.9% ± 5.3% in the control animals vs. 10.6% ± 5.3% in TTX-treated pigs) or VIP (1.7% ± 0.6% vs. 0%), and an increase in the number of FB+/TH+ neurons immunoreactive to SOM (8.8% ± 1.6% vs. 39% ± 12.8%), CB (1.8% ± 0.7% vs. 12.6% ± 2.7%), GAL (1.7% ± 0.8% vs. 10.9% ± 2.6%) or nNOS (0% vs. 1.1% ± 0.3%). The present study is the first to suggest that TTX modifies the chemical coding of CaMG neurons supplying the porcine urinary bladder.

Ghrelin-immunoreactive cells in the gastrointestinal tract of hypertensive rats.

INTRODUCTION: Ghrelin, an appetite-stimulating hormone secreted by the endocrine cells of the gastrointestinal (GI) tract, has recently been shown to affect the function of the cardiovascular system. This study aimed to assess the number and morphology of ghrelin-immunopositive (GhrIP) cells in the gastrointestinal tract of rats at different developmental phases of experimentally evoked renovascular hypertension. MATERIAL AND METHODS: The study involved 40 rats divided into two groups: control (C; n = 20) and rats with experimentally induced hypertension (EH; n = 20). The Goldblatt model of two-kidneys, one clip (2K1C) was used to induce hypertension. Renovascular hypertension was maintained for either 3 (EH1 group; n = 10) or 42 (EH2 group; n = 10) days. Paraffin sections from the cardia, corpus and pylorus of the stomach, as well as from the duodenum, jejunum, ileum and colon were processed for peroxidase immunohistochemistry. RESULTS: The number of GhrIP cells was significantly higher in the cardia and corpus of the stomach as well as the duodenum and jejunum of hypertensive rats compared to that found in the control animals. CONCLUSIONS: The increased number of GhrIP cells in the rat gastrointestinal tract after partial unilateral ligation of the renal artery suggests that renovascular hypertension may affect ghrelin secretion, which can contribute to the development of cardiovascular complications.

Morphological and neuroanatomical study of the mammary gland in the immature and mature European beaver (Castor fiber).

This study investigated general morphology and immunohistochemical properties of nerve fibres supplying the mammary gland (MG) in the European beaver. The microscopic analysis of the beaver mammary gland revealed the presence of morphological structures which are characteristic for mammals. There were no distinct differences in the morphological features of the mammary gland between the juvenile and non-pregnant mature beaver. The nerve fibres were visualized using antibodies against protein gene product 9.5 (PGP) and biologically active substances including ß-hydroxylase tyrosine (DßH), neuropeptide Y (NPY), calcitonine gene-related peptide (CGRP) and substance P (SP). The study has revealed that the MG in the juvenile and mature beaver is richly supplied with PGP-immunoreactive (PGP-IR) nerve fibres. The most abundant innervation was observed in the nipple and less numerous nerve terminals supplied the glandular tissue. Double-labelling immunohistochemistry disclosed that the majority of PGP-IR nerve fibres associated with blood vessels and smooth muscle cells in both the nipple and glandular tissue were also DßH-IR. However, these nerve terminals were less numerous in the glandular tissue than in the nipple. Most of the DßH-IR axons associated with arteries and smooth muscle cells in the entire gland also stained for NPY. Small number of DßH/NPY-IR fibres supplied veins. CGRP-IR fibres were more abundant than those expressing SP. No distinct differences in the distribution and immunohistochemical characteristic of nerve fibres were observed between the juvenile and adult animals. The distribution and immunohistochemical properties of nerve fibres supplying the gland in the beaver remind those previously described in other mammalian species.

Protective Effects of Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) Against Oxidative Stress in Zebrafish Hair Cells.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a pleiotropic neuropeptide, with known antiapoptotic functions. Our previous in vitro study has demonstrated the ameliorative role of PACAP-38 in chicken hair cells under oxidative stress conditions, but its effects on living hair cells is now yet known. Therefore, the aim of the present study was to investigate in vivo the protective role of PACAP-38 in hair cells found in zebrafish (Danio rerio) sense organs-neuromasts. To induce oxidative stress the 5-day postfertilization (dpf) zebrafish larvae were exposed to 1.5 mM H2O2 for 15 min or 1 h. This resulted in an increase in caspase-3 and p-38 MAPK level in the hair cells as well as in an impairment of the larvae basic behavior. To investigate the ameliorative role of PACAP-38, the larvae were incubated with a mixture of 1.5 mM H2O2 and 100 nM PACAP-38 following 1 h preincubation with 100 nM PACAP-38 only. PACAP-38 abilities to prevent hair cells from apoptosis were investigated. Whole-mount immunohistochemistry and confocal microscopy analyses revealed that PACAP-38 treatment decreased the cleaved caspase-3 level in the hair cells, but had no influence on p-38 MAPK. The analyses of basic locomotor activity supported the protective role of PACAP-38 by demonstrating the improvement of the fish behavior after PACAP-38 treatment. In summary, our in vivo findings demonstrate that PACAP-38 protects zebrafish hair cells from oxidative stress by attenuating oxidative stress-induced apoptosis.