Outcomes of sympathectomy and vascular bypass for digital ischaemia in connective tissue disorders.

All patients (36 hands) with connective tissue disorders who underwent periarterial sympathectomy of the hand alone or in conjunction with vascular bypass at our institution between 1995-2013 were reviewed. The durable resolution of ulcers was significantly higher in patients treated by periarterial sympathectomy and bypass than in patients treated by periarterial sympathectomy alone. Although there were more digital amputations in patients treated by periarterial sympathectomy alone, the difference was not statistically significant. Vascular bypass in conjunction with sympathectomy may be better than sympathectomy alone in patients with digital ischaemia related to connective tissue disorders. LEVEL OF EVIDENCE: IV.

Identification of the G-protein-coupled ORL1 receptor in the mouse spinal cord by [35S]-GTPgammaS binding and immunohistochemistry.

1 Although the ORL1 receptor is clearly located within the spinal cord, the functional signalling mechanism of the ORL1 receptor in the spinal cord has not been clearly documented. The present study was then to investigate the guanine nucleotide binding protein (G-protein) activation mediated through by the ORL1 receptor in the mouse spinal cord, measuring the modulation of guanosine-5'-o-(3-[35S]-thio) triphosphate ([35S]-GTPgammaS) binding by the putative endogenous ligand nociceptin, also referred as orphanin FQ. We also studied the anatomical distribution of nociceptin-like immunoreactivity and nociceptin-stimulated [35S]-GTPgammaS autoradiography in the spinal cord. 2 Immunohistochemical staining of mouse spinal cord sections revealed a dense plexus of nociceptin-like immunoreactive fibres in the superficial layers of the dorsal horn throughout the entire length of the spinal cord. In addition, networks of fibres were seen projecting from the lateral border of the dorsal horn to the lateral grey matter and around the central canal. 3 In vitro [35S]-GTPgammaS autoradiography showed high levels of nociceptin-stimulated [35S]-GTPgammaS binding in the superficial layers of the mouse dorsal horn and around the central canal, corresponding to the areas where nociceptin-like immunoreactive fibres were concentrated. 4 In [35S]-GTPgammaS membrane assay, nociceptin increased [35S]-GTPgammaS binding of mouse spinal cord membranes in a concentration-dependent and saturable manner, affording maximal stimulation of 64.1+/-2.4%. This effect was markedly inhibited by the specific ORL1 receptor antagonist [Phe1Psi (CH2-NH) Gly2] nociceptin (1 - 13) NH2. None of the mu-, delta-, and kappa-opioid and other G-protein-coupled receptor antagonists had a significant effect on basal or nociceptin-stimulated [35S]-GTPgammaS binding. 5 These findings suggest that nociceptin-containing fibres terminate in the superficial layers of the dorsal horn and the central canal and that nociceptin released in these areas may selectively stimulate the ORL1 receptor to activate G-protein. Furthermore, the unique pattern of G-protein activation in the present study provide additional evidence that nociceptin is distinct from the mu-, delta- or kappa-opioid system.

Biaxial residual stress states of plasma-sprayed hydroxyapatite coatings on titanium alloy substrate.

Biaxial residual stress states of plasma-sprayed hydroxyapatite coatings (HACs) on titanium alloy substrate as a function of plasma power, powder feed rate and coating thickness were studied by X-ray 'sin2 psi' method. The Young's modulus of hydroxyapatite (HA), required for the stress analysis, was measured from the separated free coating by three-point bending test method. It was found that the directions of principal stresses were in proximity to and perpendicular to the spraying direction. The measured Young's moduli of HACs were much lower than the theoretical value reported. The denser, well-melted HAC exhibited a higher residual stress, as compared with the less dense, poor-melting HAC. The denser coatings could be effected by higher plasma power and lower powder feed rate. Significantly, the thicker 200 microm HAC exhibited higher residual stress than that of the thinner 50 microm HAC. The implications of residual stress in HAC for biomaterials are discussed in detail.

Quantitative Autoradiography on [(35)S]TBPS Binding Sites of Gamma- Aminobutyric Acid(A) Receptors in Discrete Brain Regions of High- Alcohol-Drinking and Low-Alcohol- Drinking Rats Selectively Bred forHigh- and Low-Alcohol Preference.

It has been documented that ethanol can potentiate brain gamma-aminobutyric acid (GABA)ergic function, and there is a close link between the GABA(A) receptor complex and effects of ethanol, including reinforcement of alcohol which is a fundamental element of alcohol preference. However, it is unknown in what discrete brain regions GABA(A) receptors might be associated with alcohol preference. In the present study, [(35)S]t-butylbicyclophosphorothionate ([(35)S]TBPS) was used to localize GABA(A) receptors in high-alcohol-drinking (HAD) rats and low-alcohol-drinking (LAD) rats which were selectively bred for high and low alcohol preference, respectively. Initial qualitative observations indicated that [(35)S]TBPS binding sites were abundant in many brain areas including the cerebral cortex, hypothalamus and amygdala of HAD and LAD rats. Furthermore, the quantitative autoradiographic analysis revealed fewer [(35)S]TBPS binding sites of GABA(A) receptors in the amygdaloid complex, central medial thalamic nucleus, lateral hypothalamic nucleus and anterior hypothalamic nucleus of HAD rats than LAD rats. Collectively, this study has indicated that HAD rats selectively bred for high alcohol preference possess lower [(35)S]TBPS binding in the brain. Since lower TBPS binding has been proposed to reflect enhanced GABAergic function, as evidenced in rats with seizure or under alcohol withdrawal, the results from the present study suggest that HAD rats might have an enhanced GABAergic function. It is thus likely that enhanced GABAergic function in the brain might be related to high alcohol preference which is characteristic in HAD rats. In addition, the present result showing no difference of [(35)S]TBPS binding in the nucleus accumbens is also in agreement with a notion that [(35)S]TBPS binding may represent only a small spectrum of the GABA(A) receptor complex which is constituted of a sophisticated subunit combination whose functional compositions are still unknown. In conclusion, the present study supports the working hypothesis that GABA(A) receptors are involved in alcohol preference in HAD rats.

Analysis of saponins from black bean by electrospray ionization and fast atom bombardment tandem mass spectrometry.

Saponins from black bean (Vigna mungo L. Hepper) were analyzed using positive and negative ion fast atom bombardment mass spectrometry (FAB-MS) and liquid chromatography/mass spectrometry. Methanol was used to extract the saponins from defatted black bean, which was partially purified by extraction with n-butanol, and the extract was dialyzed with 3000 M(r) cut-off tubing. The dialyzate was analyzed using mass spectrometry. According to FAB-MS/MS, mixtures from black bean contain soyasaponin I as the predominant saponin. In addition, MS/MS analysis was performed in which the structures of saponins of black bean cotyledon were determined to be soyasaponin I, soyasaponin II, soyasaponin V, 3-O-[alpha-L-rhamnopyranosyl-(1 --> 2)-beta-D-galactopyranosyl-(1 --> 2)-beta-D-glucuronopyranosyl]complogenin (saponin A) and 3-O-[alpha-L-rhamnopyranosyl-(1 --> 2)-beta-D-glucopyranosyl-(1 --> 2)-beta-D-glucopyranosyl]oleanolic acid (saponin B). For the black bean shell and the root of black bean sprout, analysis confirmed the saponins of soyasaponin I, soyasaponin II, soyasaponin V, saponin A, saponin B, acetylsoyasaponin A(4) and soyasaponin beta(g). Moreover, all the studied saponins were found in the stem and leaves of the black bean sprouts, except soyasaponin beta(g) and acetylsoyasaponin A(4), respectively.

A rapid and sensitive radioimmunohistochemical assay for quantitation of vasopressin in discrete brain regions with an anatomical resolution.

Radioimmunoassay has become a widely used method to study different neuroactive substances from brain tissue extracts, but cannot provide anatomical resolution. Here we describe a simple and sensitive radioimmunohistochemical assay (RIHA) to quantify a peptide, vasopressin (VP), in discrete brain regions of rats with 3-day water deprivation. After decapitation, brains were removed, frozen with dry ice and cut into 14-microns cryostat sections which were then fixed with 4% paraformaldehyde in phosphate-buffered saline. After rinses, tissue sections were stored in a freezer until use. For RIHA, brain tissue sections were pre-incubated, and then incubated with rabbit vasopressin antibody (1:2000 dilution) for 24 h at room temperature. After rinses, sections were incubated with 125I-labeled goat antirabbit IgG (1:200 dilution) for 1 h. Specimens were processed for quantitative autoradiography after rinses and drying. RIHA with aid of a computer-assisted image analysis system revealed that the VP content was significantly reduced in the paraventricular hypothalamic nucleus (PVN) and supraoptic nucleus (SON) of rats with 3-day water deprivation, whereas a parallel in situ hybridization study further demonstrated that VP mRNAs in the PVN and SON were greatly increased. In summary, this experiment demonstrates that RIHA is a simple and powerful tool which is able to detect changes of VP in the hypothalamus of dehydrated rats. Combining this method with in situ hybridization to assess mRNA expression allows assessment of the functional significance of the peptide changes. In this case, dehydration depletes vasopressin and upregulates its synthesis. Therefore, the combined use of RIHA and in situ hybridization should have general applicability to evaluate the functional role of a peptide or neurotransmitter system in response to stimuli in a quantitative way with anatomical resolution.

Ultrastructural studies on catecholaminergic terminals and GABAergic neurons in nucleus tractus solitarius of the rat medulla oblongata.

Synaptogenesis of catecholamine (CA) boutons in the nucleus tractus solitarius (NTS) was compared between spontaneously hypertensive (SHR) rats and Wistar-Kyoto (WKY) rats at different ages. On the average, there were about 32 CA varicosities per 2200 microns2 area of the NTS in both SHR and WKY rats as revealed by glyoxylic acid fluorescence microscopic (FM) morphometric study. The FM analysis indicated that there were no significant changes in the CA varicosity density between SHR and WKY rats. The CA boutons were labeled with 5-hydroxydopamine and appeared to contain small granular vesicles at the electron microscopic (EM) level. A total of 1402 CA boutons were studied in a 540,000 micron2 area of the NTS. The number of CA boutons involved in synaptic contacts vs the number of total CA boutons was used to obtain synaptic frequency which was taken as an index for synaptogenesis. A reduction of approximately 18% and 14% of synaptogenesis of CA boutons was observed in the NTS of SHR rats at 4 weeks (prehypertensive stage) and 12 weeks (early hypertensive stage) of age respectively, as compared to age-matched WKY rats. No significant difference of synaptogenesis of CA neurons was found between SHR and WKY rats at 16 weeks of age, a stage in which hypertension is well established and maintained in SHR rats. These results suggest that CA neurons with fewer synaptic contacts in the NTS may play a more important role in the initiation than in the maintenance of hypertension in the SHR rats. In addition to CA terminals, there were numerous GABAergic cell bodies in the NTS which were identified by immunocytochemistry using antibodies to the GABA synthesizing enzyme, L-glutamate decarboxylase (GAD). GABAergic dendrites with GAD-positive reaction were often seen to receive several GAD-negative synapses at EM random profiles. In the text, a viewpoint is thus discussed that emphasizes that a synaptic abnormality of CA terminals with fewer synaptic contacts affecting GABAergic neurons may participate in the pathogenesis of hypertension. However, it remains to be determined as to whether or not there is a direct contact between CA boutons and GABAergic dendrites.

Characterization of monoaminergic terminals in the neostriatum of neonatal rats: an electron microscopic morphometric analysis.

Monoaminergic (MA) boutons in the neostriatum of neonatal rats were studied by using 5-hydroxydopamine. We found: (a) bouton size averaged 0.6 micron in diameter; (b) terminal density was 68 boutons per 7200 micron2 area; (c) synaptic frequency was 33.2%; and (d) MA neurons constituted 9.2% of all boutons. This population was much lower than that reported in immature neocortex where MA neurons are a major innervation.

Plasticity of catecholaminergic terminals in rat paraventricular hypothalamic nucleus after 6-hydroxydopamine lesion: an emphasis on bouton sizes and synaptic frequency.

Catecholaminergic (CA) nerve terminals in the paraventricular hypothalamic nucleus (PVN) of adult rats were studied at 4, 21, 56 and 180 days after a single injection of 6-hydroxydopamine (6-OHDA) neurotoxin into the right lateral ventricle of the brain. We previously described and quantified the extent of CA terminal sprouting in the PVN after 6-OHDA lesions. For this communication we studied parameters, specifically the bouton sizes and the synaptic frequencies of CA terminals during the renewal process, and evaluated how changes of these parameters are related to axonal sprouting. The CA boutons were identifiable in the electron microscope by exhibiting small granular vesicles (SGVs) after central administration of 5-hydroxydopamine (5-OHDA) marker. The marked CA boutons were measured and further categorized according to whether or not they were associated with distinct synaptic specializations at various post-lesion stages. The average sizes of CA boutons were strikingly similar in their diameters (1.0 micron) for both control and experimental tissues. However, CA boutons larger than 2.1 micron were rare and seen more often in the experimental tissues with 6-OHDA lesion and were sustained up to 180 days after lesions. Catecholaminergic profiles with ultrastructural features of growth cones were also seen in the PVN following the 6-OHDA lesions, indicating that there is growth activity in the PVN after 6-OHDA lesion. There were 33% of CA boutons in the PVN from the control tissues that appeared to have synaptic contacts.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcitonin gene-related peptide (CGRP) content and CGRP receptor binding sites in discrete forebrain regions of alcohol-preferring vs. -nonpreferring rats, and high alcohol-drinking vs. low alcohol-drinking rats.

This study showed that alcohol-preferring (P) rats and high alcohol-drinking (HAD) rats possess fewer calcitonin gene-related peptide (CGRP) receptor binding sites than their respective controls in the central amygdaloid nucleus (CeA) which is known to be related to anxiety. Since P and HAD rats are selectively bred for high alcohol preference, and alcohol can produce anxiolytic effect, one can postulate that P and HAD rats preferentially drink alcohol in order to obtain its anxiolytic effect. This study supports a hypothesis that deficit of CGRP receptors in the CeA of P and HAD rats may contribute to alcohol preference.

Galanin-containing neurons in the solitary nucleus and locus coeruleus of spontaneously hypertensive rats are associated with genetic hypertension.

Spontaneously hypertensive (SHR) rats contained more galanin (GAL) content and GAL mRNA in locus coeruleus (LC) at the prehypertensive, but not at the well-established hypertensive stage, than did age-matched Wistar-Kyoto (WKY) rats. However, there was also more GAL content, but not GAL mRNA, in the nucleus tractus solitarii (NTS) of SHR rats than WKY rats at both stages. This study suggests that galaninergic neurons in the LC and NTS may participate in the pathogenesis of genetic hypertension.

Increased delta, but not mu, opiate receptor binding in the medulla oblongata of Long-Evans rats following 5-day water deprivation.

Opiate receptors of the mu type were labeled with [125I]D-Ala2,N-Me-Phe4,Met-(O)5-ol-enkephalin (FK-33824). delta receptors were labeled with [125I]D-Ala2-D-Leu5-enkephalin (DADLE) in the presence of excess (N-Me-Phe3,D-Pro4)-morphiceptin (PL017). Since DADLE binds mu and delta receptor sites, and PL017 blocks mu receptors, this protocol improves specific labeling of delta receptors. Quantitative autoradiography showed that chronic dehydration causes no changes in mu receptor binding in the medulla oblongata of Long-Evans rats. However, there is increased delta receptor binding in the solitary, hypoglossal and gracilis nuclei, and the spinal nucleus of trigeminal system of dehydrated animals, suggesting that delta opiate receptors participate in the physiological response to dehydration.

The novel benzodiazepine inverse agonist RO19-4603 antagonizes ethanol motivated behaviors: neuropharmacological studies.

The novel imidazothienodiazepine inverse agonist RO19-4603 has been reported to attenuate EtOH intake in home cage drinking tests for at least 24 h post-drug administration after systemic administration. In the present study, selectively bred alcohol-preferring (P) rats were trained under a concurrent (FR4-FR4) operant schedule to press one lever for EtOH (10% v/v) and another lever for saccharin (0.05% or 0.75% g/v), then dose-response and timecourse effects of RO19-4603 were evaluated. Systemic RO19-4603 injections (0.0045-0.3 mg/kg; i.p.) profoundly reduced EtOH responding by as much as 97% of vehicle control on day 1. No effects were seen on saccharin responding except with the highest dose level (0.3 mg/kg). In a second experiment, microinjections of RO19-4603 (2-100 ng) directly into the nucleus accumbens (NA) suppressed EtOH responding on day 1 by as much as 53% of control: Control injections dorsal to the NA or ventral tegmental area did not significantly alter EtOH or saccharin responding. On day 2, rats in both experiments received no RO19-4603 treatments; however, all 7 of the i.p. doses, and all 3 of the intra-NA infusions continued to significantly suppress EtOH responding by 43-85% of vehicle control levels. In addition, i.p. injections of RO19-4603 produced a dose-dependent decrease in the slope of the cumulative record for EtOH responding, while concomitantly producing a dose-dependent increase in the slope for saccharin responding. RO19-4603's actions appear to be mediated via recognition sites at GABAA-BDZ receptors which regulate EtOH reinforcement, and not via mechanisms regulating ingestive behaviors. Based on recent in situ hybridization studies in our laboratory, we hypothesize that occupation of alpha4 containing GABAA diazepam insensitive (DI) receptors in the NA, may mediate in part, the RO19-4603 suppression of EtOH responding in EtOH-seeking P rats.

Quantitative autoradiographic study on tyrosine hydroxylase mRNA with in situ hybridization and alpha 2 adrenergic receptor binding in the locus coeruleus of the spontaneously hypertensive rat.

alpha-2 Adrenergic (A2) receptors and tyrosine hydroxylase (TH) mRNA in the locus coeruleus (LC) were studied using [125I]iodoclonidine and [35S]TH oligonucleotide probe. Spontaneously hypertensive (SHR) rats contained less TH mRNA at their prehypertensive, but not at the well-established hypertensive stage, than age-matched Wistar-Kyoto rats. Furthermore, there is an up-regulation of A2 receptors in SHR rats which is parallel to their blood pressure elevation. The present data suggest that increased A2 receptors in conjunction with TH mRNA reduction in the LC are associated with initiation, but not maintenance of genetic hypertension.

Effects of chronic dehydration on angiotensin II receptor binding in the subfornical organ, paraventricular hypothalamic nucleus and adrenal medulla of Long-Evans rats.

Angiotensin II (AII) is an important peptide known to regulate blood pressure and body fluid. In the present study we used a potent AII antagonist, 125I-(Sar1,Ile8)-AII (125I-SI-AII), to study AII receptor binding in Long-Evans rats 5 days after water deprivation. Specific structures evaluated include the subfornical organ (SFO) and adrenal gland. With quantitative autoradiography, we have found that there is an increase of 125I-SI-AII binding in the SFO, whereas there is a decrease in AII binding in the adrenal medulla. These observations suggest that central and peripheral AII target tissues are affected differently by dehydration. The increase in SI-AII binding in the SFO may indicate participation of this structure during dehydration, as angiotensin stimulation of SFO causes thirst and vasopressin release.

Stress-induced changes of norepinephrine uptake sites in the locus coeruleus of C57BL/6J and DBA/2J mice: a quantitative autoradiographic study using [3H]-tomoxetine.

Inbred C57BL/6J (C57) and DBA/2J (DBA) mice were subjected to open-field evaluation and Porsolt swim test after restraint stress. Norepinephrine (NE) uptake sites in the locus coeruleus (LC) of these inbred mice were studied by using [3H]-tomoxetine. Results showed that naive C57 mice were more active in the open field and possessed more NE uptake sites in the LC than naive DBA mice. Previous work has shown that restraint decreases open field activity in C57 mice, but not DBA mice, whereas the present study has demonstrated that, after restraint stress, C57 mice spent more time immobile than DBA mice did in the forced swim test. Furthermore, in these stressed animals, NE uptake sites in the LC were greatly increased with consistently more uptake sites in C57 mice. Collectively, results of this study and the literature suggest that enhanced NE function in the LC of C57 mice is associated with their susceptibility to stress-induced behavioral depression.

The influence of high fat diet on the fibrinolytic activity.

High fat diet containing 50% fat was given to 28 Chinese healthy volunteers for 3 days. Their mean age was 28.04 years with SD 5.53. Blood glucose, cholesterol, triglyceride (TG) and venous occlusion test (VOT) were determined before and after diet. Tissue plasminogen activator (tPA) and plasminogen activator inhibitor (PAI) were determined before and after VOT. After high-fat diet, blood glucose and cholesterol increased significantly (p = 0.031, 0.049 respectively), but other parameters did not. TPA increased significantly after VOT either before or after high-fat diet, but such increment was significantly less after high-fat diet (P < 0.05). In conclusion, the immediate effect of short-term high-fat diet included increased plasma levels of glucose and cholesterol, and impaired fibrinolytic response in stress condition.

Lower GABAA receptor binding in the amygdala and hypothalamus of spontaneously hypertensive rats.

The central GABAergic system is associated with normal blood pressure regulation, but the role of GABA receptors in genetic hypertension remains unclear. This study was conducted to investigate GABAA receptor binding in several brain regions of spontaneously hypertensive (SHR) rats during development of hypertension. GABAA receptor binding was labeled with [35S]TBPS and was assessed by quantitative autoradiography with the aid of a computer-assisted image analysis system. Densities of GABAA receptor binding sites were significantly lower in all hypothalamic and amygdaloid nuclei evaluated in 4-week-old SHR rats, when compared with their age-matched normotensive Wistar-Kyoto rats. At 12 weeks of age, GABAA receptor binding remained significantly lower in the central amygdaloid nucleus and paraventricular hypothalamic nucleus of SHR rats. Collectively, the results suggest that GABAA receptors in these nuclei are likely to be involved in the initiation and maintenance of hypertension. In conclusion, this study supports a notion that downregulation of GABAA receptor binding occurs in the hypothalamus and amygdala of SHR rats and may play a role in genetic hypertension.

Downregulation of corticotropin-releasing factor mRNA, but not vasopressin mRNA, in the paraventricular hypothalamic nucleus of rats following nutritional stress.

Stress can cause disturbance of homeostasis to result in illness. Stress can also induce various gene expression in different neuronal systems. For example, nutritional stress induced by acute food deprivation upregulates corticotropin-releasing factor (CRF) mRNA, whereas osmotic stress increases vasopressin (VP) mRNA. However, it is unknown if nutritional stress induced by chronic food deprivation has synergistic effects on CRF and VP mRNAs. We have used in situ hybridization in conjunction with quantitative autoradiography to demonstrate that nutritional stress induced by a 4-day food deprivation results in a body-weight loss with a significant decrease of CRF mRNAs, but not VP mRNAs in the paraventricular hypothalamic nucleus (PVN) of Sprague-Dawley rats. The present study has thus indicated that a chronic nutritional stress does not have synergistic effects on CRF and VP mRNAs. The decrease of CRF mRNAs is obviously related to the body-weight loss induced by food deprivation. This study thus supports a notion that the CRF, but not VP, neurons in the PVN play an important role in their neuroadaptation associated with body weight loss. Thus, it is conceivable that downregulated CRF neurons in the hypothalamus could be involved in pathogenesis of human eating disorder with severe weight loss, whereas upregulated CRF neurons could be associated with an opposite form of the eating disorder that causes obesity.

Fluorescence microscopy used in conjunction with horseradish peroxidase localization and electron microscopy for studying sympathetic nuclei of the rat spinal cord.

After application of horseradish peroxidase (HRP) to the transected cervical sympathetic trunk, labeled sympathetic neurons were seen in the intermediolateral nucleus (IML), central autonomic nucleus (CAN) and intercalated nucleus of rat spinal cords. As studied by glyoxylic acid-induced histofluorescence microscopy (FM), catecholaminergic (CA) terminals were most densely packed in the IML. There were 60 +/- 2 CA varicosities per 2,200 mu2 area in 20 mu thick sections through the IML. A combined FM/HRP method confirmed that preganglionic sympathetic neurons are heavily innervated by CA terminals. CA boutons were tagged with 5-hydroxydopamine for EM identification, and in the IML, 56% of CA boutons were seen to make synaptic contacts as compared to 60% in the CAN. It seems likely that virtually all CA boutons may form synapses but serial sections through boutons were not studied. It is inferred that preganglionic sympathetic neurons of both IML and CAN are served by CA inputs, and electron microscopy has revealed that most or all of these CA terminals transmit their signals via synapses; which may permit more precise structural sorting than non-synaptic transmission.