Local cutaneous nerve terminal and mast cell responses to manual acupuncture in acupoint LI4 area of the rats

Previous studies have shown that the effects of manual acupuncture (MA) are contributed by collagen fibers and mast cells in local acupoints, at which acupuncture stimulation causes various afferent fiber groups to be excited. However what happens in local nerve fibers and mast cells after MA remains unclear. The aim of this study was to examine the response of cutaneous nerve fibers and mast cells to MA stimulation in acupoint Hegu (LI4). The contralateral LI4 of the same rat was used as a non-stimulated control. Immnohistochemistry analysis were carried out to observe the expression of histamine (HA), serotonin (5-HT) and nociceptive neuropeptides, calcitonin gene-related peptide (CGRP) and substance P (SP), in the LI4 area. Mast cells were labeled with anti-mast cell tryptase antibody and simultaneously with HA or 5-HT primary antibodies to observe their co-expression. Our results showed that SP and CGRP were expressed more highly on the cutaneous nerve fibers of LI4 after MA stimulation than that of the control. Mast cells aggregated in close proximity to the blood vessels in intra-epidermis and dermis and some of them with degranulation in the lower dermis and subcutaneous tissue of LI4. Both mast cells and their granules appeared with HA (+) and 5-HT (+) expression at stimulated L14 sites, while a few intact mast cells with a little expression of 5-HT and HA were distributed in areas of non-stimulated L14. The results indicated that local cutaneous nerve terminals and mast cells responded to MA with higher expression of SP and CGRP in nerve fibers, as well as with aggregation and degranulation of mast cells with HA and 5-HT granules at acupoint LI4. These neuroactive substances may convey signals to certain pathways that contribute to the effects of acupuncture.     

Distribution of methionine-enkephalin in the minipig brainstem

We have studied the distribution of immunoreactive cell bodies and axons are containing methionine-enkephalin in the minipig brainstem. Immunoreactive axons were widely distributed, whereas the distribution of perikarya was less widespread. A high or moderate density of axons containing methionine-enkephalin were found from rostral to caudal levels in the substantia nigra, nucleus interpeduncularis, nucleus reticularis tegmenti pontis, nucleus dorsalis raphae, nucleus centralis raphae, nuclei dorsalis and ventralis tegmenti of Gudden, locus ceruleus, nucleus sensorius principalis nervi trigemini, nucleus cuneatus externalis, nucleus tractus solitarius, nuclei vestibularis inferior and medialis, nucleus ambiguus, nucleus olivaris inferior and in the nucleus tractus spinalis nervi trigemini. Immunoreactive perikarya were observed in the nuclei centralis and dorsalis raphae, nucleus motorius nervi trigemini, nucleus centralis superior, nucleus nervi facialis, nuclei parabrachialis medialis and lateralis, nucleus ventralis raphae, nucleus reticularis lateralis and in the formatio reticularis. We have also described the presence of perikarya containing methionine-enkephalin in the nuclei nervi abducens, ruber, nervi oculomotorius and nervi trochlearis. These results suggest that in the minipig the pentapeptide may be involved in many physiological functions (for example, proprioceptive and nociceptive information; motor, respiratory and cardiovascular mechanisms).     

Nuclear organization of some immunohistochemically identifiable neural systems in two species of the Euarchontoglires: A Lagomorph,Lepus capensis,and a Scandentia,Tupaia belangeri

The present study describes the organization of the nuclei of the cholinergic, catecholaminergic, serotonergic and orexinergic systems in the brains of two members of Euarchontoglires, Lepus capensis and Tupaia belangeri. The aim of the present study was to investigate the nuclear complement of these neural systems in comparison to previous studies on Euarchontoglires and generally with other mammalian species. Brains were coronally sectioned and immunohistochemically stained with antibodies against choline acetyltransferase, tyrosine hydroxylase, serotonin and orexin-A. The majority of nuclei revealed in the current study were similar between the species investigated and to mammals generally, but certain differences in the nuclear complement highlight potential phylogenetic interrelationships within the Euarchontoglires and across mammals. In the northern tree shrew the nucleus of the trapezoid body contained neurons immunoreactive to the choline acetyltransferase antibody with some of these neurons extending into the lamellae within the superior olivary nuclear complex (SON). The cholinergic nature of the neurons of this nucleus, and the extension of cholinergic neurons into the SON, has not been noted in any mammal studied to date. In addition, cholinergic neurons forming the medullary tegmental field were also present in the northern tree shrew. Regarding the catecholaminergic system, the cape hare presented with the rodent specific rostral dorsal midline medullary nucleus (C3), and the northern tree shrew lacked both the ventral and dorsal divisions of the anterior hypothalamic group (A15v and A15d). Both species were lacking the primate/megachiropteran specific compact portion of the locus coeruleus complex (A6c). The nuclei of the serotonergic and orexinergic systems of both species were similar to those seen across most Eutherian mammals. Our results lend support to the monophyly of the Glires, and more broadly suggest that the megachiropterans are more closely related to the primates than are any other members of Euarchontoglires studied to date.     

The centrally projecting Edinger–Westphal nucleus—I: Efferents in the rat brain

The oculomotor accessory nucleus, often referred to as the Edinger–Westphal nucleus [EW], was first identified in the 17th century. Although its most well known function is the control of pupil diameter, some controversy has arisen regarding the exact location of these preganglionic neurons. Currently, the EW is thought to consist of two different parts. The first part [termed the preganglionic EW—EWpg], which controls lens accommodation, choroidal blood flow and pupillary constriction, primarily consists of cholinergic cells that project to the ciliary ganglion. The second part [termed the centrally projecting EW—EWcp], which is involved in non-ocular functions such as feeding behavior, stress responses, addiction and pain, consists of peptidergic neurons that project to the brainstem, the spinal cord and prosencephalic regions. However, in the literature, we found few reports related to either ascending or descending projections from the EWcp that are compatible with its currently described functions. Therefore, the objective of the present study was to systematically investigate the ascending and descending projections of the EW in the rat brain. We injected the anterograde tracer biotinylated dextran amine into the EW or the retrograde tracer cholera toxin subunit B into multiple EW targets as controls. Additionally, we investigated the potential EW-mediated innervation of neuronal populations with known neurochemical signatures, such as melanin-concentrating hormone in the lateral hypothalamic area [LHA] and corticotropin-releasing factor in the central nucleus of the amygdala [CeM]. We observed anterogradely labeled fibers in the LHA, the reuniens thalamic nucleus, the oval part of the bed nucleus of the stria terminalis, the medial part of the central nucleus of the amygdala, and the zona incerta. We confirmed our EW–LHA and EW–CeM connections using retrograde tracers. We also observed moderate EW-mediated innervation of the paraventricular nucleus of the hypothalamus and the posterior hypothalamus. Our findings provide anatomical bases for previously unrecognized roles of the EW in the modulation of several physiologic systems.     

Histological studies of neuroprotective effects of Curcuma longa Linn. on neuronal loss induced by dexamethasone treatment in the rat hippocampus

Long term exposure to dexamethasone (Dx) is associated with brain damage especially in the hippocampus via the oxidative stress pathway. Previously, an ethanolic extract from Curcuma longa Linn. (CL) containing the curcumin constituent has been reported to produce antioxidant effects. However, its neuroprotective property on brain histology has remained unexplored. This study has examined the effects of a CL extract on the densities of cresyl violet positive neurons and glial fibrillary acidic protein immunoreactive (GFAP-ir) astrocytes in the hippocampus of Dx treated male rats. It showed that 21days of Dx treatment (0.5 mg/kg, i.p. once daily) significantly reduced the densities of cresyl violet positive neurons in the sub-areas CA1, CA3 and the dentate gyrus, but not in the CA2 area. However, CL pretreatment (100 mg/kg, p.o.) was found to significantly restore neuronal densities in the CA1 and dentate gyrus. In addition, Dx treatment also significantly decreased the densities of the GFAP-ir astrocytes in the sub-areas CA1, CA3 and the dentate gyrus. However, CL pretreatment (100 mg/kg, p.o.) failed to protect the loss of astrocytes in these sub-areas. These findings confirm the neuroprotective effects of the CL extract and indicate that the cause of astrocyte loss might be partially reduced by a non-oxidative mechanism. Moreover, the detection of neuronal and glial densities was suitable method to study brain damage and the effects of treatment.     

Mutations in rpoB gene and rifabutin susceptibility of multidrug-resistant Mycobacterium tuberculosis strains isolated in Australia.

Control of tuberculosis, the single largest killer among the infectious diseases, has been threatened by the emergence of multidrug-resistant Mycobacterium tuberculosis (MDRTB) infection due to the limited treatment options. Rifampicin (RIF) resistance is considered as a marker for MDRTB. The aim of this study was the detection of rpoB gene mutations and rifabutin resistance in MDRTB strains recently isolated in Australia by a line probe assay (INNO-LiPA Rif. TB, Innogenetics). Rifabutin and RIF susceptibility of 20 MDRTB and 16 RIF-sensitive M. tuberculosis complex clinical isolates were studied. The overall concordance of the line probe assay (LiPA) with phenotypic RIF susceptibility test was 96%. Seven distinct nucleotide substitutions were identified in 21 of 22 RIF-resistant isolates of diverse geographical origins, but in none of the RIF-sensitive strains. The majority (71%) of mutations occurred in the 526-533 codons and were associated with resistance to rifabutin and RIF. Of the RIF-resistant MDRTB strains, 18% appeared to be rifabutin-sensitive and produced delta S2 and delta S3 INNO-LiPA patterns. We conclude that amino acid substitutions at Asp516 and Ser522 in the rpoB gene in RIF-resistant M. tuberculosis predict rifabutin susceptibility for MDRTB. Use of the LiPA for RIF and rifabutin resistance may facilitate the rapid response required to limit the extent and severity of MDRTB transmission and infection.     

Effect of mobile telephony on blood-brain barrier permeability in the fetal mouse brain

AIMS: To study the effect of mobile telephone exposure on blood-brain barrier (BBB) permeability in the immature brain. METHODS: Using a purpose-designed exposure system at 900 MHz, pregnant mice were given a single, far-field, whole body exposure at a specific absorption rate of 4 W/kg for 60 min/day from day 1 to day 19 of gestation. Pregnant control mice were sham-exposed or freely mobile in a cage without further restraint and a positive control group with cadmium-induced BBB damage was also included. Immediately prior to parturition on gestational day 19, fetal heads were collected, fixed in Bouin's fixative and paraffin embedded. Disruption of BBB integrity was detected immunohistochemically using endogenous albumin as a vascular tracer in cerebral cortex, thalamus, basal ganglia, hippocampus, cerebellum, midbrain and medulla. RESULTS: No albumin extravasation was found in exposed or control brains. CONCLUSION: In this animal model, whole of gestation exposure to global system for mobile communication-like radiofrequency fields did not produce any increase in vascular permeability in the fetal brain regions studied using endogenous albumin as a light microscopic immunohistochemical marker.     

Nuclear organization of cholinergic,catecholaminergic, serotonergic and orexinergic systems in the brain of the Tasmanian devil (Sarcophilus harrisii)

This study investigated the nuclear organization of four immunohistochemically identifiable neural systems (cholinergic, catecholaminergic, serotonergic and orexinergic) within the brains of three male Tasmanian devils (Sarcophilus harrisii), which had a mean brain mass of 11.6 g. We found that the nuclei generally observed for these systems in other mammalian brains were present in the brain of the Tasmanian devil. Despite this, specific differences in the nuclear organization of the cholinergic, catecholaminergic and serotonergic systems appear to carry a phylogenetic signal. In the cholinergic system, only the dorsal hypothalamic cholinergic nucleus could be observed, while an extra dorsal subdivision of the laterodorsal tegmental nucleus and cholinergic neurons within the gelatinous layer of the caudal spinal trigeminal nucleus were observed. Within the catecholaminergic system the A4 nucleus of the locus coeruleus complex was absent, as was the caudal ventrolateral serotonergic group of the serotonergic system. The organization of the orexinergic system was similar to that seen in many mammals previously studied. Overall, while showing strong similarities to the organization of these systems in other mammals, the specific differences observed in the Tasmanian devil reveal either order specific, or class specific, features of these systems. Further studies will reveal the extent of change in the nuclear organization of these systems in marsupials and how these potential changes may affect functionality.     

Complex phenotypes in the haemoglobinopathies: recommendations on screening and DNA testing

This document considers a number of scenarios involving complex haemoglobinopathies and provides 28 recommendations at both the clinical and laboratory levels on how these should be managed.     

Broadsheet. Number 50: Diagnosis of human cytomegalovirus infection and disease.

Human cytomegalovirus (CMV) remains an important cause of illness in immunocompromised individuals and the most common viral cause of congenital malformation. The tests available for diagnosis of CMV include serology, antigen detection, virus culture, tissue histopathology and nucleic acid detection. The diagnosis of CMV remains difficult because of the issues of virus latency, virus infection versus clinical disease and virus reactivation. The tests available and the use of these tests are undergoing significant changes. This Broadsheet presents a review of these tests, particularly in the diagnosis of congenital infection and infection in pregnant women and immunocompromised individuals.     

Orexinergic innervation of urocortin1 and cocaine and amphetamine regulated transcript neurons in the midbrain centrally projecting Edinger–Westphal nucleus

Orexin is a neuropeptide that has been implicated in several processes, such as induction of appetite, arousal and alertness and sleep/wake regulation. Multiple lines of evidence also suggest that orexin is involved in the stress response. When orexin is administered intracerebroventricular it activates the hypothalamic pituitary adrenal (HPA)-axis, which is the main regulator of the stress response. The HPA-axis is not the only player in the stress response evidence suggests that urocortin 1 (Ucn1), a member of the corticotropin releasing factor (CRF) neuropeptide family, also plays an important role in the stress response adaptation. Ucn1 is primarily synthetized in the centrally projecting Edinger–Westphal nucleus (EWcp), which also receives dense innervation by orexin terminals. In this study we tested the hypothesis that orexin would directly shape the response of EWcp-Ucn1 neurons to acute cold stress. To test this hypothesis, we first assessed whether orexinergic axon terminals would innervate EWcp-Ucn1/CART neurons, and next we exposed orexin deficient (orexin-KO) male mice and their male wild-type (WT) littermates to acute cold stress for 2 h. We also assessed stress-associated changes in plasma corticosterone (CORT), as well as the activation of Ucn1/CART neurons in the EWcp nucleus. We found that orexin immunoreactive axon terminals were juxtaposed to EWcp-Ucn1/CART neurons, which also expressed orexin receptor 1 mRNA. Furthermore, acute stress strongly activated the EWcp-Ucn1/CART neurons and increased plasma CORT in both WT littermates and orexin-KO mice, however no genotype effect was found on these indices. Taken together our data show that orexin in general is not involved in the animal's acute stress response (plasma CORT) and it does not play a direct role in shaping the response of EWcp-Ucn1 neurons to acute stress either.     

The use of coroner's autopsy reports to validate the use of targeted swabbing rather than tissue collection for rapid confirmation of virological causes of sudden death in the community

Background and objectivesIn this study, coroner's autopsy reports were used to validate results obtained from respiratory virus screening of swabs rather than tissue collected during autopsy in cases of adult death of unknown cause.Study designCoroner's autopsy samples collected for respiratory virus screening between October 2010 and February 2011, were identified. Autopsy reports were requested from cases positive for a virus. Each report was reviewed to correlate findings at autopsy with the virology result and to determine whether the virus found was listed as a contributing factor in the death.ResultsSixty-four coroner's autopsy cases were identified and a respiratory virus was found in 25 cases. Influenza A(H1N1)pdm09 virus was found most frequently, then RSV and influenza B with a dual influenza A and B infection and a parainfluenza type 1. Where multiple sites were swabbed, the virus was detected in all sites. Autopsy reports for 12 cases were obtained each reporting findings consistent with respiratory infection. Influenza A was always listed as a contributing factor in the death whereas RSV was listed once and influenza B was omitted in one case. The quality of the reports was variable and full histology was less likely to be performed in the elderly.ConclusionsWhile coroner's reports supported the use of swabbing rather than tissue collection, the lack of consistency and omission of the virology findings as contributing factors to death means that the burden of viruses on mortality statistics will remain under-estimated particularly in the elderly.     

Projections from the rat cuneiform nucleus to the A7, A6 (locus coeruleus), and A5 pontine noradrenergic cell groups

Stimulation of neurons in the cuneiform nucleus (CnF) produces antinociception and cardiovascular responses that could be mediated, in part, by noradrenergic neurons that innervate the spinal cord dorsal horn. The present study determined the projections of neurons in the CnF to the pontine noradrenergic neurons in the A5, A6 (locus coeruleus), and A7 cell groups that are known to project to the spinal cord. Injections of the anterograde tracer, biotinylated dextran amine in the CnF of Sasco Sprague-Dawley rats labeled axons located near noradrenergic neurons that were visualized by processing tissue sections for tyrosine hydroxylase-immunoreactivity. Anterogradely labeled axons were more dense on the side ipsilateral to the BDA deposit. Both A7 and A5 cell groups received dense projections from neurons in the CnF, whereas locus coeruleus received only a sparse projection. Highly varicose anterogradely labeled axons from the CnF were found in close apposition to dendrites and somata of tyrosine hydroxylase-immunoreactive neurons in pontine tegmentum. Although definitive evidence for direct pathways from CnF neurons to the pontine noradrenergic cell groups requires ultrastructural analysis, the results of the present studies provide presumptive evidence of direct projections from neurons in the CnF to the pontine noradrenergic neurons of the A7, locus coeruleus, and A5 cell groups. These results support the suggestion that the analgesia and cardiovascular responses produced by stimulation of neurons in the CnF may be mediated, in part, by pontine noradrenergic neurons.     

Laboratory testing of anticoagulants: the present and the future

This review provides an update on laboratory testing and monitoring for existing and emerging anticoagulants, starting with an overview of haemostasis and the routine coagulation tests currently employed within most haemostasis laboratories, including the prothrombin time (PT)/international normalised ratio (INR) and the activated partial thromboplastin time (APTT). Current anticoagulant therapy and laboratory monitoring is then discussed in terms of benefits and limitations, followed by a similar brief discussion of the new and emerging anticoagulants. The main focus, however, is laboratory testing related to vitamin K antagonists, heparin, lepirudin and the new agents dabigatran etexilate and rivaroxaban. Although the newer agents do not require laboratory monitoring, laboratory testing will occasionally be required, and pathology laboratories should become proactive in developing appropriate strategies. The tests most likely to fulfill this role are the ecarin clotting time (or chromogenic alternatives), and the chromogenic anti-Xa assay. Nevertheless, the dilute Russell viper venom time (dRVVT) assay may provide another alternative, and existing routine tests are also likely to be utilised for the foreseeable future, potentially also for laboratory testing of the new anticoagulants, albeit perhaps in modified form.     

Subcellular localization and activation of ADAM proteases in the context of FasL shedding in T lymphocytes

The “A Disintegrin And Metalloproteinases” (ADAMs) form a subgroup of the metzincin endopeptidases. Proteolytically active members of this protein family act as sheddases and govern key processes in development and inflammation by regulating cell surface expression and release of cytokines, growth factors, adhesion molecules and their receptors. In T lymphocytes, ADAM10 sheds the death factor Fas Ligand (FasL) and thereby regulates T cell activation, death and effector function. Although FasL shedding by ADAM10 was confirmed in several studies, its regulation is still poorly defined. We recently reported that ADAM10 is highly abundant on T cells whereas its close relative ADAM17 is expressed at low levels and transiently appears at the cell surface upon stimulation. Since FasL is also stored intracellularly and brought to the plasma membrane upon stimulation, we addressed where the death factor gets exposed to ADAM proteases. We report for the first time that both ADAM10 and ADAM17 are associated with FasL-containing secretory lysosomes. Moreover, we demonstrate that TCR/CD3/CD28-stimulation induces a partial positioning of both proteases and FasL to lipid rafts and only the activation-induced raft-positioning results in FasL processing. TCR/CD3/CD28-induced FasL proteolysis is markedly affected by reducing both ADAM10 and ADAM17 protein levels, indicating that in human T cells also ADAM17 is implicated in FasL processing. Since FasL shedding is affected by cholesterol depletion and by inhibition of Src kinases or palmitoylation, we conclude that it requires mobilization and co-positioning of ADAM proteases in lipid raft-like platforms associated with an activation of raft-associated Src-family kinases.     

Molecular biomarkers in malignant mesothelioma: state of the art

Malignant mesothelioma (MM) is an aggressive tumour affecting the mesothelial surfaces of the pleural and peritoneal cavities and, rarely, the pericardium and the tunica vaginalis testis. Despite a ban of asbestos in many industrialised nations, the present high incidence of MM is expected to continue, due to the long latency period between first asbestos exposure and occurrence of disease, making it an important health issue for the future. The diagnosis of MM can be difficult, both from a clinical and pathological perspective. It is not unusual for patients to undergo several medical investigations without definitive diagnosis early in their course of illness. Understandably, there is intense interest in the discovery of markers that can be assessed in pleural effusions, histological specimens, and serum to assist with the difficult early diagnosis of MM. Considering the primary aetiological role of asbestos, there is theoretically an easily identifiable target population for screening with a biomarker with adequate sensitivity and specificity or with a combination of biomarkers. In this review we focus on biomarkers that have been examined in the setting of either early diagnosis of MM in symptomatic patients or screening of asbestos-exposed individuals.     

Cytotoxicity evaluation and antioxidant enzyme expression related to heavy metals found in tuna by-products meal: An in vitro study in human and rat liver cell lines

Heavy metals can accumulate in organisms via various pathways, including respiration, adsorption and ingestion. They are known to generate free radicals and induce oxidative and/or nitrosative stress with depletion of anti-oxidants. Tuna by-product meal (TBM) is rich in proteins and can, therefore, offer an attractive protein source for animals. This study was undertaken to assess the effects of metals present in TBM, namely cadmium (Cd), lead (Pb), and mercury (Hg), separately or in combination with oxidative stress, on cell viability. Three cell models: rat liver FTO2B, human hepatoma HepG2, and human hepatic WRL-68, were used. Cell viability was determined following exposure to various concentrations of the metals. Two antioxidant genes, catalase (CAT) and superoxide dismutase (SOD), were measured to obtain a better understanding of oxidative stress-associated gene expression. Among the metals present in TBM, only Cd at a concentration of 30 μM was noted to exhibit cytotoxic effects. This cytotoxicity was even more pronounced after co-stimulation with H₂O₂, used to mimic systemic oxidative stress. At non-toxic concentrations, Hg and Pb were noted to aggravate oxidative stress toxicity. The results further revealed that exposure to Cd, Pb, and a co-stimulation of H₂O₂ with Hg resulted in the increased expression of antioxidant gene SOD. A risk assessment of toxic contaminants in TBM indicated that food safety objectives should consider the human health impacts of foods derived from animals fed on contaminated meal and that much care should be taken when TBM is used in animal diet.