Salt chamber treatment is ineffective in treating eosinophilic inflammation in asthma.

BACKGROUND: We have shown that salt chamber treatment reduces airway hyper-responsiveness as an add-on therapy in adult asthmatics on inhaled corticosteroids. METHODS: We assessed whether this effect is due to the suppression of eosinophilic airway inflammation. Thirty-nine adult asthmatics on inhaled corticosteroids were randomized to receive active salt chamber treatment with low-salt treatment 6.6 mg/m(3) (n = 14), high-salt treatment 10.8 mg/m(3) (n = 15) or placebo 0.3 mg/m(3) (n = 10) 10 times in a 2 weeks' period in a double-blind manner. RESULTS: The level of induced sputum eosinophilic cationic protein µg/l, was 3070 before and 4651 after the low-salt treatment period, on average. In the high-salt treatment group, it was 12 192 µg/l vs 11 803 and in the placebo group 3942 vs 4144, respectively. Salt chamber treatment had no effect on sputum eosinophil or neutrophil cell numbers. CONCLUSIONS: The reduction in hyper-responsiveness observed in the previous study is probably not due to the effect on eosinophilic inflammation.

Can age-based estimates of weight be safely used when resuscitating children?

BACKGROUND: Prescribing medication appropriate to a child's bodily dimensions is fundamental to paediatric emergency medicine. Mathematical formulae are frequently used in clinical practice to estimate children's weights. In 1995 the UK's paediatric reference data, describing age-related changes in bodily proportions (both weight and height), were updated and published. This study assesses the validity of using mathematical estimates, age-based estimates or length-based estimates of weight (the latter both compiled from this reference data) by comparison with actual physical measurements recorded in a paediatric clinic setting. METHODS: A prospective study was carried out in a paediatric outpatient setting recording age, weight and height for statistical comparison with these three possible methods. RESULTS: 544 children aged 0-11 years were recruited, with mean (SD) age, weight and height of 5.3 (2.9) years, 21.4 (10) kg and 108 (22) cm, respectively. CONCLUSIONS: Both length-based and age-based estimates of weight outperformed the currently accepted "gold standard" mathematical estimate when applied to children up to 11 years of age (approximately 35 kg). Length-based estimates were statistically superior, but the physical limitations and technical constraints posed when attempting to accurately measure a child's length in emergency environments may favour the simplicity of using the child's age against tables of growth chart reference data to provide an estimate of their weight.

Prehospital paediatric emergency care: paediatric triage.

The practice of triage was conceived during the Napoleonic wars, with the aim of salvaging those soldiers whose injuries were readily treatable, returning them to the battlefield at the earliest opportunity. Literally, the word triage means "to sieve" or "to sort" (French trier), and those earlier battlefield principles have been refined and expanded to now encompass trauma and medical emergencies, with triage practiced in prehospital and hospital settings. To address the anatomical, physiological and developmental differences encountered when dealing with children, specific paediatric triage systems have also been developed, and this article discusses their merits.

NADPH diaphorase cells in the mammalian inner retina.

The distribution of the enzyme NADPH diaphorase was examined histochemically in the retina of the rat, rabbit, cat, owl monkey, squirrel monkey, rhesus macaque, and human being. In all species tested the enzyme was concentrated in cells 10-12 microns in diameter at the vitread margin of the inner nuclear layer. The population was sparse (less than 2,000 cells/rat retina). In several species additional minor populations were observed. While a clear function for NADPH diaphorase has yet to be described, an abundance of the enzyme characterizes a similar subpopulation of retinal cells in a wide variety of mammals.

NADPH diaphorase histochemistry in the macaque striate cortex.

The distribution of the enzyme dihydronicotinamide adenine dinucleotide phosphate (NADPH) diaphorase was examined in the striate cortex of the rhesus monkey. The pattern of activity in the neuropil matched that of cytochrome oxidase in adjacent sections and the enzymes were similarly modulated by monocular deprivation. Scattered individual cells were also intensely positive for NADPH diaphorase. Labelled cells were most common in the white matter and layers 2 and 3; they were least common in layers 4 and 5. Diaphorase cells were morphologically diverse, but no pyramidal or spiny cells were labelled. Labelled cells often had multiple varicose processes, which extended laterally for over 1 mm. Although the function of this enzyme is unknown, the morphology and distribution of the diaphorase-positive cells resembles published reports of cortical cells containing somatostatin, avian pancreatic polypeptide, and neuropeptide Y-like immunoreactivity, and NADPH diaphorase is colocalized with these substances in the rodent striatum (S.R. Vincent, O. Johansson, T. Hökfelt, L. Skirboll, R.P. Elde, L. Terenius, J. Kimmel, and M. Goldstein, J. Comp. Neurol. 217:252-263, '83).

Effects of age on nerve fibers in the rhesus monkey optic nerve.

During normal aging there is a reduction in white matter volume in the cerebral hemispheres and structural abnormalities in myelin in some parts of the central nervous system, but whether nerve fibers are lost with age and whether the myelin changes are ubiquitous is not known. Studying the optic nerve, which is a circumscribed bundle of nerve fibers, offers an opportunity to gain further insight into the effects of normal aging on white matter. The present study examined the optic nerves from young (4-10 years) and old (27-33 years) rhesus monkeys using light and electron microscopy. These nerves had been perfused transcardially to obtain optimal preservation of the tissue. Varying degrees of degeneration were encountered in all the optic nerves from the old monkeys. The changes included myelin abnormalities, similar to those reported in other parts of the central nervous system; the presence of degenerating axons and their sheaths; changes in neuroglial cells; and thickening of the trabeculae of connective tissue in the nerve. The total number of nerve fibers was reduced from an average of 1.6 x 10(6) in the young optic nerves to as few as 4 x 10(5) in one old monkey, and with one exception in all of the old optic nerves the packing density of nerve fibers was less than in any of the young optic nerves. The degenerative changes were most marked in those optic nerves that contained the fewest nerve fibers.

Shape and distribution of an unusual retinal neuron.

Rabbit retinas were exposed to exogenous indoleamines and fixed with mixed aldehydes. The indoleamines were accumulated by two types of amacrine cell and by an unusual cell (type 3) that branches widely in both plexiform layers. The type 3 cells were studied after immunohistochemistry, photooxidation of the fluorescent label, or injection with Lucifer Yellow. Their cell bodies are located at the scleral margin of the inner nuclear layer. The cells' arbors in the outer plexiform layer range from 800 to 1,500 microns in diameter. A descending process crosses the inner nuclear layer and branches in layer 5 of the inner plexiform layer. The arbor in the inner retina can exceed 500 microns in diameter. The distribution of type 3 cells was mapped in a series of retinal whole mounts. The number of type 3 cells ranged from 58 to 270 in different retinas. In two retinas from a single animal, however, it was virtually identical. Type 3 cells are concentrated in the region ventral to the visual streak, so that large areas of the retina are not covered by any type 3 cell. Because of their incomplete retinal coverage and variable number from animal to animal, the type 3 cells appear to be developmental anomalies. Paradoxically, their generation must be precisely controlled because of the numerical symmetry between an individual animal's two eyes.

Visual topography of V2 in the macaque.

The representation of the visual field in the area adjacent to striate cortex was mapped with multiunit electrodes in the macaque. The animals were immobilized and anesthetized and in each animal 30 to 40 electrode penetrations were typically made over several recording sessions. This area, V2, contains a topographically organized representation of the contralateral visual field up to an eccentricity of at least 80 degrees. The representation of the vertical meridian is adjacent to that in striate cortex (V1) and forms the posterior border of V2. The representation of the horizontal meridian in V2 forms the anterior border of V2 and is split so that the representation of the lower visual field is located dorsally and that of the upper field ventrally. As in V1, the representation of the central visual field is magnified relative to that of the periphery. The area of V2 is slightly smaller than that of V1. At a given eccentricity, receptive field size in V2 is larger than in V1. The myeloarchitecture of V2 is distinguishable from that of the surrounding cortex. The location of V2 corresponds, at least approximately, to that of cytoarchitectonic Area OB. V2 is bordered anteriorly by several other areas containing representations of the visual field.

Sequence and expression of glutamic acid decarboxylase isoforms in the developing zebrafish.

We describe the isolation two glutamic acid decarboxylase (GAD) cDNAs from zebrafish with over 84% identity to human GAD65 and GAD67. In situ hybridization studies revealed that both GAD65 and GAD67 were expressed in the early zebrafish embryo during the period of axonogenesis, suggesting a role for GABA prior to synapse formation. Both GAD genes were detected in the telencephalon, in the nucleus of the medial longitudinal fasciculus in the midbrain, and at the border regions of the rhombomeres in the rostral hindbrain. In the caudal hindbrain, only GAD67 was detected (in neurons with large-caliber axons). In the spinal cord, both GAD genes were detected in dorsal longitudinal neurons, commissural secondary ascending neurons, ventral longitudinal neurons, and Kolmer-Agduhr neurons. Immunohistochemistry for gamma-aminobutyric acid (GABA) revealed that GABA is produced at all sites of GAD expression, including the novel cells in the caudal hindbrain. These results are discussed in the context of the hindbrain circuitry that supports the escape response. We conclude that fish, like mammals, have two GAD genes. The zebrafish GAD65 and GAD67 are present in identified neurons in the forebrain, midbrain, hindbrain, and spinal cord, and they catalyze the production of GABA in the developing embryo.

The development of GABA immunoreactivity in the retina of the zebrafish (Brachydanio rerio).

The goal of this study was to determine the pattern of gamma-aminobutyric acid (GABA) expression in the retina and optic nerve of the zebrafish (Brachydanio rerio) during embryonic development. Zebrafish embryos were fixed at intervals between 1 and 4 days postfertilization, and semithin plastic sections were prepared for postembedding immunocytochemistry with antisera against GABA. Sections were also prepared from several adult zebrafish eyes for comparison. GABA immunoreactivity first appeared in the optic nerve at 2 days postfertilization, and by 2.5 days the inner nuclear layer (INL), inner plexiform layer (IPL), retinal ganglion cell layer, and optic nerve were all positive for GABA. The GABA expression in the retinal ganglion cell layer and optic nerve was transient, however, and these structures were largely unlabeled by 4 days postfertilization. The pattern of GABA immunoreactivity at 4 days resembled that seen in the adult zebrafish: A large population of presumptive amacrine cells was labeled at the base of the INL, and the IPL was positive for GABA, as were occasional cells in the ganglion cell layer. Horizontal cells, particularly at the retinal margins, were also GABA positive beginning at about 3 days postfertilization. The transient expression of GABA in retinal ganglion cells and their axons during the period when synaptic contacts are being established both within the retina and between the retina and central targets suggests that GABA may have a role in the development of this system, in addition to serving as a classical neurotransmitter.

Connections of indoleamine-accumulating cells in the rabbit retina.

To study the connections of the neurons of the rabbit retina that accumulate indoleamines, we injected 5,7-dihydroxytryptamine into the vitreous body. It accumulated within a subset of amacrine cells and could be visualized there by aldehyde-induced fluorescence. The fluorescent labeling was photo-converted to an insoluble, osmiophilic product by irradiation in the presence of diaminobenzidine, and the tissue was examined by electron microscopy. Preservation of the structure of the tissue after photoconversion was satisfactory and the dendrites of the indoleamine-accumulating cells could easily be identified. They form a dense plexus near the junction of the inner plexiform and ganglion cell layers, where they exhibit large synaptic endings that occupy a substantial fraction of the surface of rod bipolar terminals. The dendrites of the indoleamine-accumulating cells receive input from rod bipolars at dyad synapses, where the other postsynaptic partner is a dendrite of a narrow-field, bistratified amacrine cell; in addition, they receive amacrine cell input throughout the inner plexiform layer. The only outputs we observed are reciprocal synapses onto the rod bipolar endings. Thus, these amacrine cells appear to exert an important effect on the transmission of scotopic information through the retina.

A new enzyme marker for striatal compartmentalization: NADPH diaphorase activity in the caudate nucleus and putamen of the cat.

The distribution of dihydronicotinamide adenine dinucleotide phosphate diaphorase (NADPH diaphorase) was studied by enzyme histochemistry in the striatum of the adult cat. Neurons and neuropil expressing NADPH diaphorase activity were found throughout the striatum. The diaphorase-positive neurons formed a sparse population of medium-sized cells. In the caudate nucleus they were recognized by antisera against somatostatin 14, somatostatin 28(1-12), neuropeptide Y and avian pancreatic polypeptide. The diaphorase activity of the striatal neuropil was characterized by a modular organization that was particularly distinct in the caudate nucleus. This organization was analyzed by comparing the patterns of diaphorase staining with the distribution of acetylcholinesterase activity in adjacent sections. The NADPH diaphorase activity was found to be dense in the acetylcholinesterase-rich matrix of the caudate nucleus, but weak in the acetylcholinesterase-poor compartments known as striosomes. Because of the colocalization of perikaryal NADPH diaphorase activity and somatostatinlike immunoreactivity, a comparison was also made between the distribution of diaphorase staining and immunostaining for somatostatinlike peptide in the striatal neuropil. Both observed striosomal ordering, so that the acetylcholinesterase-poor zones detected in adjoining sections corresponded to regions of low somatostatinlike immunoreactivity as well as low NADPH diaphorase staining. In some regions striosomes were more clearly delineated in the stains for diaphorase and somatostatinlike suggest that NADPH diaphorase may be a sensitive marker for the somatostatinergic neuropil as well as the somatostatinergic perikarya of the striatum, and that this enzyme could prove valuable in attempts to differentiate the processes of intrinsic somatostatin-containing fibers from any extrinsic somatostatin afferents that may exist.

The distribution of neurotrophin receptor TrkC-like immunoreactive fibers and varicosities in the rhesus monkey brain.

The distribution of immunoreactivity for the neurotrophin receptor tyrosine kinase TrkC was examined in the brain of the adult rhesus monkey. TrkC-like immunoreactivity was widespread and consisted primarily of varicose fibers. The most dense populations of fibers were in the basal forebrain (in the cholinergic cell groups Ch1, Ch2 and Ch4), in the raphé complex throughout its rostrocaudal extent, and in the locus coeruleus. Other fibers were present in the thalamus, hypothalamus, central gray matter of the midbrain, dorsal midline of the brainstem and the cerebral cortex. The only neuronal cell bodies with consistent labeling were located in the lateral hypothalamus. Purkinje cells in the cerebellum showed variable labeling. Specific labeling of varicosities and cell bodies was abolished by omission of the primary antiserum or by preabsorption with the TrkC peptide antigen. We conclude that TrkC-like immunoreactivity can be detected in a wide variety of subcortical locations in the adult rhesus monkey. Labeling was particularly prominent in the vicinity of the major cholinergic, serotonergic and adrenergic nuclei, known from other studies to be vulnerable in the ageing brain. This suggests that the ligand for TrkC, neurotrophin-3, may persist as a survival factor for critical neurons into adulthood.

Photoconversion of some fluorescent markers to a diaminobenzidine product.

Retinal whole mounts, brain sections, and astrocyte cultures were labeled with various fluorescent markers. Tissues or cells were then irradiated by light in the presence of diaminobenzidine. Irradiation initiated a reaction in which specific fluorescent labeling was replaced by an insoluble diaminobenzidine product. The diaminobenzidine product is more stable than the original fluorescent labeling and can be processed for electron microscopy. In some cases, the reaction product reveals cellular detail that cannot be resolved in the fluorescent labeling. The 10 fluorescent markers tested have widely differing structures, span a broad range of wavelengths, and label several different cellular elements. The photoconversion reaction was successful with all markers and tissues tested.

Carbon-11-NNC 112: a radioligand for PET examination of striatal and neocortical D1-dopamine receptors.

UNLABELLED: The aim of this work was to explore the potential of a selective D1-dopamine receptor antagonist as a new radioligand for PET examination of striatal and neocortical D1-dopamine receptors. METHODS: The active (+)- and inactive (-)-enantiomers of [11C]NNC 112 were radiolabeled using the N-methylation approach and were examined by PET in cynomolgus monkeys and healthy men. Metabolite levels in plasma were measured by gradient high-performance liquid chromatography (HPLC). RESULTS: N-methylation of the corresponding desmethyl precursors with [11C]methyl triflate gave high total radiochemical yield (50%-60%) and specific radioactivity (110 GBq/micromol). (+)-[11C]NNC 112 binding in cynomolgus monkeys was 5.77+/-0.31 and 2.36+/-0.14 times higher in the striatum and neocortex, respectively, than in the cerebellum at a transient equilibrium that appeared 40-50 min after injection. The binding of (+)-[11C]NNC 112 is stereoselective, because the brain distribution of the inactive (-)-enantiomer was on an equally low level for all brain regions. Displacement and pretreatment experiments using unlabeled SCH 23390 and ketanserin confirms that (+)-[11C]NNC 112 binds specifically and reversibly to D1-dopamine receptors. The radioactivity ratios of the striatum, frontal cortex and nucleus accumbens to the cerebellum were 3.8-4.0, 1.7-2.0 and 2.8-3.1, respectively, at a transient equilibrium that appeared 40-50 min after injection in four healthy human subjects. Linear graphical analysis gave distribution volume ratios of 3.9 and 1.5 in the putamen and frontal cortex, respectively. The fraction of the total radioactivity in human plasma representing unchanged (+)-[11C]NNC 112 was 85% at 5 min and 25% at 75 min after injection. CONCLUSION: (+)-[11C]NNC 112 should be a useful PET radioligand for quantitative examination of not only striatal but neocortical D1-dopamine receptors in man.

The distribution of hexokinase compared to cytochrome oxidase and acetylcholinesterase in the somatosensory cortex and the superior colliculus of the rat.

The distribution of hexokinase, a general glycolytic enzyme, was compared to that of cytochrome oxidase and acetylcholinesterase (AChE) in the somatosensory cortex and the superior colliculus of the rat. The vibrissal barrel fields of the adult rat contain high hexokinase and cytochrome oxidase activity and low AChE activity. In the superior colliculus, hexokinase activity was highest in cell layers and discrete foci of intense activity were observed in the deep grey layer. This distribution was different from that of both cytochrome oxidase and AChE in this structure.

The development of neurotrophin receptor Trk immunoreactivity in the retina of the zebrafish (Brachydanio rerio).

The purpose of this study was to examine the cellular distribution of the Trk family of neurotrophin receptors in the retina and optic nerve of the zebrafish (Brachydanio rerio) during embryonic development. Semithin sections from zebrafish retinae were examined immunohistochemically for the presence of Trk polypeptides using commercially available antisera that cross-react with the fish. Cross-reactivity was confirmed by Western blot. Trk polypeptides were detected at about 1 day of age on the surfaces of retinal neuroblasts and faint Trk immunoreactivity was observed in the primordial optic nerve at 1.5 days. By 2 days the optic nerve was clearly positive for Trk and at 2.5 days Trk immunoreactivity was found in the outer plexiform, inner nuclear, inner plexiform and ganglion cell layers, as well as in the optic nerve. At 3 days and 4 days the location of Trk immunoreactivity was unchanged but by 4 days it had diminished in intensity. In the adult zebrafish retina Trk immunoreactivity was found in the same locations as in the embryonic fish, as well as in a population of cells in the middle of the inner nuclear layer and in photoreceptors. We conclude that Trk neurotrophin receptors are present in the zebrafish eye during development and that their persistence in the adult may support the continuous neural reorganization that accompanies the growth of the eye in the fish.

Disruption of a taste familiarity effect by novel exteroceptive stimulation.

Preexposed or familiar tastes are normally retarded relative to novel tastes in acquiring aversive properties as a consequence of being paired with an illness-inducing event. Four experiments are reported which demonstrate that such a taste familiarity effect can be significantly disrupted by exposing the animal to novel exteroceptive stimulation just prior to conditioning with the familiar taste. Implications of this finding for theories of taste aversion learning are discussed, and two potential theoretical accounts of the data are suggested.

Use of PET and the radioligand [carbonyl-(11)C]WAY-100635 in psychotropic drug development.

Positron-emission tomography (PET) provides potential in neuropsychiatric drug development by expanding knowledge of drug action in the living human brain and reducing time consumption and costs. The 5-hydroxytryptamine(1A) (5-HT(1A)) receptor is of central interest as a target for the treatment of anxiety, depression, and schizophrenia. Research on the clinical significance of the 5-HT(1A) receptor now benefits from the highly selective radioligand [carbonyl-(11)C]WAY-100635 (WAY) for quantitative determination of 5-HT(1A) receptors in the primate and human brain in vivo using PET. In this paper, three studies are reviewed to demonstrate the suitability of WAY as radioligand for quantification of central 5-HT(1A) receptors in brain and as an applicable tool for drug development. In the first study a monkey model was used to characterize WAY binding. It was confirmed that the reference ligand 8-OH-DPAT and psychoactive drugs such as buspirone and pindolol occupies 5-HT(1A) receptors in the primate brain. Pindolol is an beta-adrenoreceptor antagonist with a high affinity to 5-HT(1A) receptors. This drug has been suggested in combination with selective serotonin reuptake inhibitors for the treatment of depression and was given to healthy males in the second study. Pindolol induced a marked inhibition of central 5-HT(1A) receptors as calculated by the ratio-analysis method and simplified reference tissue model, 2 h after administration of 10 mg as a single oral dose. This observation suggests that pindolol may have a role for the suggested potentiation of selective serotonin reuptake inhibitor treatment of depression. The third study was on robalzotan (NAD-299), a recently developed 5-HT(1A) receptor antagonist and putative drug with implications for the treatment of depression. In the cynomolgus monkey brain, robalzotan in the dose range 2-100 microg/kg IV occupied 5-HT(1A) receptors in a dose-dependent and saturable manner with a maximal calculated occupancy of 70-80%. The relationship between robalzotan plasma concentration and 5-HT(1A) receptor occupancy could be described by a hyperbolic function that was used to guide the selection of appropriate doses in man. In a subsequent PET study of robalzotan binding to 5-HT(1A) receptors in the living human brain, similar results have been replicated recently. These studies reviewed here illustrate and corroborate that quantitative neuroimaging of receptor binding has potential for the evaluation and dose finding of new central nervous system drugs.

Carbon-11 pb-12: an attempt to visualize the dopamine d(4) receptor in the primate brain with positron emission tomography.

The dopamine D(4) receptor (D(4)R) is expressed in low density in various extrastriatal brain regions. This receptor subtype is discussed in relation to the pathophysiology and treatment of schizophrenia but no selective positron emission tomography (PET) ligand is available to date to study the distribution in vivo. The arylpiperazine derivative N-[2-[4-(4-chlorophenyl)piperazin-1-yl]ethyl]-3-methoxybenzamide (PB-12) is a novel, high-affinity ( K(i)=0.040 nM) and selective D(4)R ligand. We radiolabeled PB-12 with carbon-11 (t(1/2) 20.4 min) by O-methylation of the corresponding desmethyl analogue N-[2-[4-(4-chlorophenyl)piperazin-1-yl]ethyl]-3-hydroxybenzamide (LM-190) with [(11)C]methyl triflate. Derivative LM-190 was prepared by condensing 3-hydroxybenzoic acid with the appropriate amine. For the radiolabeling, the incorporation yield was >90% and the total synthesis time including high performance liquid chromatography (HPLC) purification was about 35 min. The specific radioactivity of [(11)C]PB-12 at time of injection was 67-118 GBq x micromol(-1). PET studies in a cynomolgus monkey showed a high uptake and widespread distribution of radioactivity in the brain, including the neocortex and thalamus. About 40% of total radioactivity in plasma represented unchanged radioligand at 60 min after injection as determined by HPLC. Pretreatment with the D(4)R ligand 3-[[4-(4-chlorophenyl)piperazin-1-yl]methyl]-1H-pyrollo[2,3-b]pyridine (L-745,870) prior to radioligand injection failed to demonstrate receptor-specific binding in the monkey brain. Furthermore, the brain radioactivity distribution was left unaffected by pretreating with unlabeled PB-12. This failure to detect a D(4)R-specific signal may be related to a very low density of the D(4)R in primate brain, insufficient binding affinity of the radioligand, and a high background of nonspecific binding. It can be concluded from these findings that [(11)C]PB-12 is not suitable to visualize the D(4)R in the primate brain with PET.