Immunohistochemical properties of motoneurons supplying the trapezius muscle in the rat

Immunohistochemical properties of motoneurons supplying the trapezius muscle in the rat Combined retrograde tracing (using fluorescent tracer Fast blue) and double-labelling immunofluorescence were used to study the distribution and immunohistochemical characteristics of neurons projecting to the trapezius muscle in mature male rats (n=9). As revealed by retrograde tracing, Fast blue-positive (FB+) neurons were located within the ambiguous nucleus and accessory nucleus of the grey matter of the spinal cord. Immunohistochemistry revealed that nearly all the neurons were cholinergic in nature [choline acetyltransferase (ChAT)-positive]. Retrogradely labelled neurons displayed also immunoreactivities to calcitonin gene-related peptide (CGRP; approximately 60% of FB+ neurons), nitric oxide synthase (NOS; 50%), substance P (SP; 35%), Leu5-Enkephalin (LEnk; 10%) and vasoactive intestinal polypeptide (VIP; 5%). The analysis of double-stained tissue sections revealed that all CGRP-, VIP- and LEnk-immunoreactive FB+ perikarya were simultaneously ChAT-positive. The vast majority of the neurons expressing SP- or NOS-immunoreactivity were also cholinergic in nature; however, solitary somata were ChAT-negative. FB+ perikarya were surrounded by numerous varicose nerve fibres (often forming basket-like structures) immunoreactive to LEnk or SP. They were also associated with some CGRP-, NOS- and neuropeptide Y-positive nerve terminals.

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Immunohistochemical characterization of efferent neurons innervating the oviduct in the pig located in the sympathetic chain ganglia

Veterinární Medicína ◽

10.17221/5809-vetmed ◽

2012 ◽

Vol 47 (No. 4) ◽

pp. 85-91

Author(s):

K. Czaja

Keyword(s):

Nerve Cells ◽

Sympathetic Chain ◽

Double Labelling ◽

Retrograde Labelling ◽

Calcitonin Gene ◽

Ovum Transport ◽

Efferent Neurons ◽

Oxide Synthase ◽

Labelling Immunofluorescence

The present study was aimed at disclosing the pattern(s) of putative co-incidence of tyrosine hydroxylase (TH), dopamine -hydroxylase (DH), neuropeptide Y (NPY), substance P (SP), calcitonin gene-related peptide (CGRP) and nitric oxide synthase (NOS) within the porcine “oviductal” efferent neurons using combined retrograde tracing and double-labelling immunohistochemistry. The fluorescent retrograde tracer Fast Blue (FB) was injected into the wall of the ampullar (n = 5) and isthmal (n = 5) part of the organ in ten sexually immature female pigs. After a survival period of three weeks sympathetic chain ganglia (SCG) were collected. 10 µm-thick cryostat sections of the ganglia were examined for the presence of FB-positive (FB+) nerve cells under the fluorescent microscope. Tracered neurons were processed for double-labelling immunofluorescence according to the method of Wessendorf and Elde. Retrograde labelling revealed a population of “oviductal” efferent neurons located in the thoracic (T) and lumbar (L) SCG at the level of T14 to L5. Double-labelling immunofluorescence allowed several subpopulations of the studied perikarya to be distinguished. The largest one consisted of TH+/DH+ (immunopositive) nerve cells. The moderate number of FB+ nerve cells expressed TH/NPY- immunoreactivity (IR). The tracered neurons did not show SP, CGRP and NOS immunoreactivity. Because identically coded nerve fibres have been observed within the wall of the porcine oviduct it can be assumed that TH+/DH+ or TH+/NPY+ neurons are involved in the control the oviductal tonus and ovum transport.

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Immunohistochemical characteristics and distribution of neurons in the intramural ganglia supplying the urinary bladder in the male pig

Polish Journal of Veterinary Sciences ◽

10.2478/pjvs-2013-0090 ◽

2013 ◽

Vol 16 (4) ◽

pp. 629-638 ◽

Cited By ~ 2

Author(s):

Z. Pidsudko

Keyword(s):

Urinary Bladder ◽

Large Population ◽

Double Labelling ◽

Chemical Coding ◽

Visceral Peritoneum ◽

Calcitonin Gene ◽

Oxide Synthase ◽

Cryostat Sections ◽

Labelling Immunofluorescence ◽

Intramural Ganglia

Abstract This study investigated the distribution and chemical coding of neurons in intramural ganglia of the urinary bladder trigone (UBT-IG) and cervix (UBC-IG) in the male pig using combined retrograde tracing and double-labelling immunohistochemistry. Additionally, immunoblotting was used to confirm the presence of marker enzymes for main populations of autonomic neurons. Retrograde fluorescent tracer Fast Blue (FB) was injected into the wall of both the left and right side of the bladder trigone, cervix and apex during laparotomy performed under thiopental anaesthesia. Twelve μm-thick cryostat sections were processed for double-labelling immunofluorescence with antibodies against tyrosine hydroxylase (TH), dopamine β-hydroxylase (DBH), neuropeptide Y (NPY), somatostatin (SOM), galanin (GAL), vasoactive intestinal polypeptide (VIP), nitric oxide synthase (NOS), calcitonin gene-related peptide (CGRP), substance P (SP) and vesicular acetylcholine transporter (VAChT). UBT-IG and UBC-IG neurons in both parts of the organ formed characteristic clusters (from few to tens of neuronal cells) found under visceral peritoneum or in the outer muscular layer. Immunohistochemistry revealed several subpopulations in UBT-IG and UBC-IG neurons, namely noradrenergic (ca. 76% and 76%), cholinergic (ca. 22% and 20%), non-adrenergic/non-cholinergic nerve cells (ca. 1.5% and 3.8%), NPY- (ca. 66% and 58%), SOM- (ca. 39% and 39 %), VIP- (ca. 5% and 0%) and NOS- immunoreactive (IR) (ca. 1.5% and 3.8%), respectively. Immunoblotting using antibodies to TH and VAChT showed the presence of studied proteins as revealed by the presence of protein bands of the correct molecular weight. This study has revealed a relatively large population of differently coded UBT- and UBC- IG neurons, which constitute an important element of the complex neuroendocrine system involved in the regulation of the male urogenital organs function.

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Distribution pattern and chemical coding of neurons of the sympathetic chain ganglia supplying the descending colon in the pig

Acta Veterinaria Hungarica ◽

10.1556/avet.58.2010.2.5 ◽

2010 ◽

Vol 58 (2) ◽

pp. 189-198 ◽

Cited By ~ 6

Author(s):

Cezary Skobowiat ◽

Jarosław Calka ◽

Krzysztof Wasowicz ◽

Mariusz Majewski

Keyword(s):

Great Majority ◽

Retrograde Tracing ◽

Sympathetic Chain ◽

Double Labelling ◽

Chemical Coding ◽

Pituitary Adenylate Cyclase ◽

Descending Colon ◽

Oxide Synthase ◽

Ventral Area ◽

Labelling Immunofluorescence

Sympathetic chain ganglia (SChG) neurons projecting to the descending colon of the pig were studied by means of retrograde tracing (Fast Blue, FB) and double-labelling immunofluorescence methods. FB was injected into the gut wall and after three weeks survival time the animals were transcardially perfused with paraformaldehyde and the bilateral sympathetic trunks were collected. The FBpositive neurons were localised only in the lumbar (L 1 –L 5 ) ganglia of the sympathetic trunk and appeared either as small (30–50 μm in diameter) round-shaped perikarya forming clusters localised in caudal-ventral area or, rarely, as bigger (50–80 μm) and dispersed solitary irregular perikarya. Immunohistochemical staining revealed the catecholaminergic (tyrosine hydroxylase-/dopamine β-hydroxylase-immunoreactive) character of the great majority of FB-positive neurons which preferentially co-expressed neuropeptide Y. In addition, none of the FB-positive perikarya was immunopositive to galanin, somatostatin, choline acetyltransferase, vasoactive intestinal peptide, pituitary adenylate cyclase-activating peptide, leu 5 -enkephalin, nitric oxide synthase, substance P and calcitonin-generelated peptide.

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Immunohistochemical characterisation of neurons in the mandibular ganglion and nerve fibres supplying the porcine mandibular gland

Veterinární Medicína ◽

10.17221/221/2015-vetmed ◽

2017 ◽

Vol 61 (No. 7) ◽

pp. 361-373 ◽

Cited By ~ 1

Author(s):

M. Klimczuk ◽

P. Podlasz ◽

W. Sienkiewicz ◽

A. Franke-Radowiecka ◽

A. Dudek ◽

...

Keyword(s):

Mandibular Gland ◽

Double Labelling ◽

Nerve Terminals ◽

Rt Pcr ◽

Nerve Fibres ◽

Chemical Coding ◽

Pcr Product ◽

Calcitonin Gene ◽

Oxide Synthase ◽

Cryostat Sections

The present study was designed to investigate the chemical coding of neurons in the mandibular ganglion (MGn) and nerve fibres supplying the porcine mandibular gland (MGl) with the use of immunofluorescence and RT-PCR. The cryostat sections from MGn and MGl were processed for double-labelling immunohistochemistry using antisera against vesicular acetylcholine transporter (VAChT), choline acetyltransferase (ChAT), dopamine β-hydroxylase (DβH), neuronal nitric oxide synthase (nNOS), vasoactive intestinal polypeptide (VIP), neuropeptide Y (NPY), galanin (GAL), substance P (SP) and calcitonin gene-related peptide (CGRP). The MGl was found to be richly supplied by VAChT-positive nerve fibres that surrounded intra- and interlobular salivary ducts. A large number of VAChT-immunoreactive (VAChT-IR) nerve terminals were also observed around acini. Many periductal and periacinar nerve fibres stained positive for DβH. Immunoreactivity to GAL, NPY or VIP was observed in an intermediate number of nerve terminals which were associated with both salivary ducts and acini. Double-immunostaining revealed that in MGn nearly all neurons stained positive for VAChT/ChAT (98.45 ± 0.59%, mean ± SEM) and nNOS (99.71 ± 0.18%). An intermediate number of the nerve cell bodies displayed immunoreactivity to NPY or VIP (18.67 ± 0.52% and 8.11 ± 0.36%, respectively). Single GAL-IR and CGRP-positive neurons were also observed. RT-PCR revealed the presence of transcripts of ChAT, VAChT, nNOS, NPY, VIP and GAL. For SP and DβH very weak signals were observed. RT-PCR with primers targeting CGRP did not generate any PCR product.

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Characterisation of cocaine- and amphetamine- regulated transcript-like immunoreactive (CART-LI) enteric neurons in the porcine small intestine

Acta Veterinaria Hungarica ◽

10.1556/avet.2012.032 ◽

2012 ◽

Vol 60 (3) ◽

pp. 371-381 ◽

Cited By ~ 20

Author(s):

Joanna Wojtkiewicz ◽

Sławomir Gonkowski ◽

Marek Bladowski ◽

Mariusz Majewski

Keyword(s):

Nitric Oxide ◽

Small Intestine ◽

The Other ◽

Enteric Neurons ◽

Double Labelling ◽

Immunofluorescence Technique ◽

Submucous Plexus ◽

Oxide Synthase ◽

First Time ◽

Labelling Immunofluorescence

The aim of this study was to investigate the distribution and the number of cocaine- and amphetamine-regulated transcript-like immunoreactive (CART-LI) neurons and the co-localisation of CART with substance P (SP), somatostatin (SOM), nitric oxide synthase (NOS) and vasoactive intestinal polypeptide (VIP) within the enteric nervous system (ENS) in the porcine small intestine. Accordingly, the myenteric plexus (MP), outer submucous plexus (OSP) and inner submucous plexus (ISP) of the small intestine (duodenum, jejunum and ileum) were studied by double-labelling immunofluorescence technique. CART-LI neurons were observed in all gut fragments and all types of intramural plexuses studied and amounted from 0.2 ± 0.1% in the ISP of ileum to 22.4 ± 2.4% in the MP of this segment. The co-localisation of CART and NOS or/and VIP was observed depending on the segment of the gut and the complexity of the intramural plexus. On the other hand, during this study the co-localisation of CART and SOM or/and SP was not observed. The present study, for the first time, presents a detailed description of the CART distribution pattern and co-localisation with other neuromodulators within the ENS of the porcine small intestine.

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Neurochemical Plasticity of nNOS-, VIP- and CART-Immunoreactive Neurons Following Prolonged Acetylsalicylic Acid Supplementation in the Porcine Jejunum

International Journal of Molecular Sciences ◽

10.3390/ijms21062157 ◽

2020 ◽

Vol 21 (6) ◽

pp. 2157

Author(s):

Dominika Rząp ◽

Marta Czajkowska ◽

Jarosław Całka

Keyword(s):

Nitric Oxide ◽

Nervous System ◽

Acetylsalicylic Acid ◽

Neuronal Plasticity ◽

Adaptive Response ◽

Neuronal Nitric Oxide Synthase ◽

Double Labelling ◽

Neurochemical Plasticity ◽

Oxide Synthase ◽

Labelling Immunofluorescence

Aspirin, also known as acetylsalicylic acid (ASA), is a commonly used anti-inflammatory drug that has analgesic and antipyretic properties. The side effects are well known, however, knowledge concerning its influence on gastric and intestinal innervation is limited. The enteric nervous system (ENS) innervates the whole gastrointestinal tract (GIT) and is comprised of more than one hundred million neurons. The capacity of neurons to adapt to microenvironmental influences, termed as an enteric neuronal plasticity, is an essential adaptive response to various pathological stimuli. Therefore, the goal of the present study was to determine the influence of prolonged ASA supplementation on the immunolocalization of neuronal nitric oxide synthase (nNOS), vasoactive intestinal peptide (VIP) and cocaine- and amphetamine- regulated transcript peptide (CART) in the porcine jejunum. The experiment was performed on 8 Pietrain × Duroc immature gilts. Using routine double-labelling immunofluorescence, we revealed that the ENS nerve cells underwent adaptive changes in response to the induced inflammation, which was manifested by upregulated or downregulated expression of the studied neurotransmitters. Our results suggest the participation of nNOS, VIP and CART in the development of inflammation and may form the basis for further neuro-gastroenterological research.

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Distribution and immunohistochemical properties of autonomic neurons supplying the ovine hip joint capsule

Veterinární Medicína ◽

10.17221/61/2017-vetmed ◽

2018 ◽

Vol 63 (No. 6) ◽

pp. 261-270

Author(s):

W. Sienkiewicz ◽

A. Dudek ◽

A. Chroszcz ◽

M. Janeczek ◽

J. Kaleczyc

Keyword(s):

Hip Joint ◽

Retrograde Tracing ◽

Joint Capsule ◽

Sympathetic Chain ◽

Double Labelling ◽

Nerve Fibres ◽

Fast Blue ◽

Mesenteric Ganglion ◽

Caudal Mesenteric Ganglion ◽

Autonomic Neurons

Combined retrograde tracing and double labelling immunohistochemistry were applied to study the distribution and chemical coding of autonomic neurons projecting to the ovine hip joint capsule. As revealed by retrograde tracing, fast blue-positive autonomic neurons supplying the lateral side of the hip joint capsule and the medial side of the hip joint capsule were located within the lumbar and sacral of the ipsilateral sympathetic chain ganglia and within the caudal mesenteric ganglion. Immunohistochemistry revealed that nearly all (sympathetic chain ganglia: 96% and caudal mesenteric ganglion: 98.8%) the neurons were adrenergic in nature (positive for dopamine β-hydroxylase). Many retrogradely labelled neurons also displayed immunoreactivity to neuropeptide Y (approximately 34% of fast blue-positive neurons within caudal mesenteric ganglion and sympathetic chain ganglia). Populations of Met-Enk+ (20%) and Leu-Enk+ (6%) neurons were present only in the sympathetic chain ganglia while within caudal mesenteric ganglion no enkephalinergic-labelled neurons were noted. Only a small population (2.2%) of hip joint capsule-projecting neurons were Gal-IR and they were observed only within the caudal mesenteric ganglion. No cholinergic neurons involved in the innervation of the hip joint capsule were found. However, fast blue-positive nerve cell bodies were surrounded by numerous cholinergic nerve fibres often forming basket-like formations. Single Gal+ nerve fibres were found in the intraganglionic connective tissue. Substance P-positive or calcitonin gene-related peptide-positive intraganglionic nerve terminals were very numerous and formed “baskets” surrounding fast blue-positive perikarya within sympathetic chain ganglias and caudal mesenteric ganglion.

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Aspirin Administration Affects Neurochemical Characterization of Substance P-Like Immunoreactive (SP-LI) Nodose Ganglia Neurons Supplying the Porcine Stomach

BioMed Research International ◽

10.1155/2020/1049179 ◽

2020 ◽

Vol 2020 ◽

pp. 1-9

Author(s):

Liliana Rytel ◽

Jarosław Całka

Keyword(s):

Substance P ◽

Control Group ◽

Double Labelling ◽

Large White ◽

Nodose Ganglia ◽

Calcitonin Gene ◽

Mucosal Layer ◽

Inflammatory Processes ◽

Oxide Synthase

Background. Acetylsalicylic acid (ASA) is a commonly used anti-inflammatory, antipyretic, and analgesic drug, which has many side effects on the gastric mucosal layer. Despite this, knowledge concerning the influence of ASA on neuronal cells supplying the stomach is very scanty. Methods. This investigation was performed on ten immature gilts of the Large White Polish race divided into two groups (five animals in each): a control group and animals which were treated with ASA. The retrograde neuronal tracer Fast Blue (FB) was injected into the prepyloric region of the stomach in all animals. ASA was then given orally to the experimental (ASA) group of gilts from the seventh day after FB injection to the 27th day of the experiment. After this period, all animals were euthanized. Immediately after euthanasia, nodose ganglia (NG) were collected and subjected to a standard double-labelling immunofluorescence technique using antibodies directed toward substance P (SP) and other selected neuronal factors, such as galanin (GAL), neuronal isoform of nitric oxide synthase (nNOS), vasoactive intestinal polypeptide (VIP), and calcitonin gene-related peptide (CGRP). Key Results. The obtained results show that SP-LI neurons located in NG supplying the porcine stomach were also immunoreactive to all the above-mentioned neuronal factors. Moreover, ASA administration caused an increase in the degree of colocalization of SP with other neuronal active substances, and the most visible changes concerned the number of neurons simultaneously immunoreactive to SP and CGRP. Conclusions and Inferences. These observations indicate that the population of SP-LI neurons supplying the stomach is not homogeneous and may undergo changes after ASA administration. These changes are probably connected with inflammatory processes and/or neuroprotective reactions although their exact mechanisms remain unknown.

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Distribution and chemical coding patterns of cocaine- and amphetamine-regulated transcript-like immunoreactive (CART-LI) neurons in the enteric nervous system of the porcine stomach cardia

Polish Journal of Veterinary Sciences ◽

10.1515/pjvs-2015-0067 ◽

2015 ◽

Vol 18 (3) ◽

pp. 515-522 ◽

Cited By ~ 6

Author(s):

W. Rękawek ◽

P. Sobiech ◽

S. Gonkowski ◽

K. Żarczyńska ◽

A. Snarska ◽

...

Keyword(s):

Nervous System ◽

Enteric Nervous System ◽

Neuronal Nitric Oxide Synthase ◽

Nerve Fibers ◽

Vesicular Acetylcholine Transporter ◽

Double Labelling ◽

Dense Network ◽

Chemical Coding ◽

Oxide Synthase ◽

Labelling Immunofluorescence

Abstract The aim of this study was to determine the presence of cocaine- and amphetamine-regulated transcript-like immunoreactive (CART-LI) neurons and co-localisation of CART with vesicular acetylcholine transporter (VAChT), neuronal nitric oxide synthase (n-NOS), vasoactive intestinal polypeptide (VIP), substance P (SP) and leu-enkephalin (LENK) in the enteric nervous system of the porcine gastric cardia by using a double-labelling immunofluorescence technique. CART-LI neurons were observed in the myenteric plexus (18.2±2.6%). A dense network of CART-LI nerve fibers was mainly observed in the muscular layer. CART showed co-localization mainly with VAChT, n-NOS, VIP and to a lesser degree with LENK and SP. Distribution of CART and its co-localization with other neurotransmitters suggest that this peptide plays an important role in gastric motility in the pig.

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Abstract 15295: Molecular Mechanism of Chronic Sildenafil-Caused Regression of Heart Failure: Effects on the Express of Cardiac SR Ca2+-ATPase, Subtype of β-Adrenergic Receptors and Nitric Oxide Synthase Isoforms

Circulation ◽

10.1161/circ.130.suppl_2.15295 ◽

2014 ◽

Vol 130 (suppl_2) ◽

Author(s):

Heng-Jie Cheng ◽

Tiankai Li ◽

Che Ping Cheng

Keyword(s):

Nitric Oxide ◽

Heart Failure ◽

Nitric Oxide Synthase ◽

Adrenergic Receptors ◽

Left Ventricular ◽

Male Rats ◽

Protein Levels ◽

Myocyte Function ◽

Oxide Synthase ◽

Β Adrenergic Receptors

Background: Sildenafil (SIL), a selective inhibitor of PDE5 has been shown to exert profound beneficial effects in heart failure (HF). Recently we further found that SIL caused regression of cardiac dysfunction in a rat model with isoproterenol (ISO)-induced progressive HF. However, the molecular basis is unclear. We hypothesized that reversal of HF-induced detrimental alterations on the expressions of cardiac SR Ca 2+ -ATPase (SERCA2a), β-adrenergic receptors (AR) and nitric oxide synthase (NOS) isoforms by SIL may play a key role for its salutary role in HF. Methods: Left ventricular (LV) and myocyte function and the protein levels of myocyte β 1 - and β 3 - AR, SERCA2a, phospholamban (PLB) and three NOS were simultaneously evaluated in 3 groups of male rats (6/group): HF , 3 months (M) after receiving ISO (170 mg/kg sq for 2 days); HF/SIL , 2 M after receiving ISO, SIL (70 μg/kg/day sq via mini pump) was initiated and given for 1 M; and Controls (C). Results: Compared with controls, ISO-treated rats progressed to severe HF at 3 M after ISO followed by significantly decreased LV contractility (E ES , HF: 0.7 vs C: 1.2 mmHg/μl) and slowed LV relaxation, reductions in the peak velocity of myocyte shortening (77 vs 136 μm/sec), relengthening (62 vs 104 μm/sec) and [Ca 2+ ] iT (0.15 vs 0.24) accompanied by a diminished myocyte inotropic response to β-AR agonist, ISO (10 -8 M). These abnormalities were associated with concomitant significant decreases in myocyte protein levels of β 1 -AR (0.23 vs 0.64), SERCA2a (0.46 vs 0.80), PLB Ser16 /PLB ratio (0.24 vs 0.40) and eNOS (0.28 vs 0.46), but significantly increases in protein levels of β 3 -AR (0.29 vs 0.10) and iNOS (0.18 vs 0.08) with relatively unchanged nNOS. Chronic SIL prevented the HF-induced decreases in LV and myocyte contraction, relaxation, peak [Ca 2+ ] iT , and restored normal myocyte contractile response to ISO stimulation. With SIL, protein levels of myocyte β 1 - and β 3 -AR, SERCA2a were restored close to control values, but eNOS was significantly elevated than controls (0.77). Conclusions: Chronic SIL prevents HF-caused downregulation of cardiac β 1 -AR and reverse contrast changes between iNOS and β 3 -AR with SERCA 2a and eNOS expression, leading to the preservation of LV and myocyte function, [Ca 2+ ] iT , and β-adrenergic reserve.

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