Effect and Mechanism of Si-Miao-Yong-An on Vasa Vasorum Remodeling in ApoE−/− Mice with Atherosclerosis Vulnerable Plague

Objective: To observe the effect of Si-Miao-Yong-An (SMYA) on atherosclerosis (AS) vulnerable plaques, and to further explore the mechanism by vasa vasorum (VV) angiogenesis and maturation as an entry point.Methods: SPF-class healthy male ApoE−/− mice were randomized into model group, simvastatin group and SMYA group, and C57BL/6 mice were used as the control group. After 8 weeks of intervention, the pathological morphology of plaque was observed by HE staining; the VV density in plaque and aortic adventitia were observed by immunohistochemistry; VV maturation was measured by double-labelling immunofluorescence; the critical proteins of HIF-1α-Apelin/APJ and Ang-1/Tie signal pathways were detected by western blotting.Results: SMYA decreased the plaque area and the ratio of plaque to lumen area; increased the minimum thickness of fibrous cap and its effect was greater than simvastatin. SMYA suppressed the VV neovascularization; promoted smooth muscle cells recruitment and VV maturation, which maintained plaque stability; its effect was obviously superior to simvastatin. SMYA deceased the expression of HIF-1α, Apelin, APJ, Phospho-MEK1/2 (Ser217/221), Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204), Phospho-p70 S6 Kinase (Thr421/Ser424), Ang-2 and Tie-2; it also increased the expression of Ang-1, Phospho-Akt (Ser473), Phospho-FOXO1 (Ser256) and Survivin.Conclusions: SMYA can decrease the AS plaque area in ApoE−/− mice, suppress the VV neovascularization and promote the VV maturation, and stabilize AS vulnerable plaque. The mechanism could be regulating the HIF-1α-Apelin/APJ and Ang-1/Tie signal pathways.

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

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.

Changes in Somatostatin-Like Immunoreactivity in the Sympathetic Neurons Projecting to the Prepyloric Area of the Porcine Stomach Induced by Selected Pathological Conditions

The aim of the present study was to define changes in the expression of somatostatin (SOM) in the sympathetic perikarya innervating the porcine stomach prepyloric area during acetylsalicylic-acid-induced gastritis (ASA) and experimentally induced hyperacidity (HCL) and following partial stomach resection (RES). On day 1, the stomachs were injected with neuronal retrograde tracer Fast Blue (FB). Animals in the ASA group were given acetylsalicylic acid orally for 21 days. On the 22nd day after FB injection, partial stomach resection was performed in RES animals. On day 23, HCL animals were intragastrically given 5 ml/kg of body weight of a 0.25 M aqueous solution of hydrochloric acid. On day 28, all pigs were euthanized. Then, 14-μm thick cryostat sections of the coeliac-superior mesenteric ganglion (CSMG) complexes were processed for routine double-labelling immunofluorescence. All pathological conditions studied resulted in upregulation of SOM-like (SOM-LI) immunoreactivity (from14.97±1.57% in control group to33.72±4.39% in the ASA group, to39.02±3.65% in the RES group, and to29.63±0.85% in the HCL group). The present studies showed that altered expression of SOM occurs in sympathetic neurons supplying the prepyloric area of the porcine stomach during adaptation to various pathological insults.

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

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.

Somatostatin-like immunoreactive primary sensory neurons supplying the porcine adrenal glands in physiological conditions and after adrenalectomy

Retrograde neuronal tracing, using fast blue, in combination with a single-labelling immunofluorescence technique, was applied to determine whether somatostatin (SOM) participates in sensory innervating of the porcine adrenal glands in physiological conditions and after adrenalectomy. In control animals, SOM-like immunoreactive neurons comprised 7.0 ± 0.7% of adrenal gland-projecting cells in dorsal root ganglia (DRG) at neuromeres Th6-7 and 6.5 ± 1.2% at neuromeres Th12-14. After adrenalectomy the percentage of SOM-positive DRG cells considerably increased and attained the level of 44.7 ± 2.5% at neuromeres Th6-7 and 36.6 ± 1.7% at neuromeres Th12-14. The obtained results demonstrate that SOM is not only a neuromediator within sensory neurones supplying the porcine adrenal glands, but also suggest the role of this substance during repairing processes within the nervous system after adrenalectomy.

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

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.

Pituitary adenylate cyclase activating peptide-27 – like immunoreactive nerve fibers in the mucosal layer of canine gastrointestinal tract in physiology and during inflammatory bowel disease

Changes in the density of mucosal pituitary adenylate cyclase activating peptide-27 -like immunoreactive (PACAP-27 - LI) nerve fibers within various parts of the canine gastrointestinal (GI) tract during inflammatory bowel disease (IBD) were investigated. The distribution of nerves were studied, using a single-labelling immunofluorescence technique, in the mucosal layer of canine stomach, duodenum, jejunum, and descending colon. Canine IBD caused an increase in the density of PACAP- 27-LI mucosal nerves in all studied parts of GI tract. The results suggest that PACAP in the nervous system may be involved in pathological processes during IBD.

Immunohistochemical characterization of neurons in the vestibular ganglion (scarpa’s ganglion) of the pig

The study was carried out on three 4-month old female pigs. All the animals were deeply anesthetized and transcardially perfused with 4% buffered paraformaldehyde (pH 7.4). Vestibular ganglia (VG) were collected and processed for double-labelling immunofluorescence method. The preparations were examined under the Zeiss LSM 710 confocal microscope equipped with adequate filter blocks. Neurons forming VG were round or oval in shape with a round nucleus in the center. The majority of them (58%) were medium (M) (31-50 μm in diameter) while 28 % and 14% were small (S) (up to 30 μm in diameter) or large (L) (above 50 μm in diameter) in size, respectively. Double-labeling immunofluorescence revealed that VG neurons stained for CGRP (approx. 81%; among them 70.5%, 26.2% and 3.3% were M, S and L in size, respectively), VACHT (57%; 63% M, 24% S, 13% L), Met-Enk (25%; 60% M, 12% S, 28% L), VIP (20%; 88% M, 6% S, L), NPY (15%; 67% M, 20% S, 13% L), GAL (15%; 74% M, 21% S, 5% L), SP (12%; 69% M, 25% S, 6% L) and NOS-positive (12%; 50% S, 50% M). The most abundant populations of intraganglionic nerve fibers were those which stained for CGRP or Met-Enk, whereas only single SP- or NOS-positive nerve terminals were observed.

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

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.

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

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.