Evaluation of post-exposure prophylaxis practices to improve the cost-effectiveness of rabies control in human cases potentially exposed to rabies in southern Bhutan.

BACKGROUND: Rabies is endemic in southern Bhutan, associated with 1-2 human deaths and high post exposure prophylaxis (PEP) costs annually. Evaluation of clinicians' management of human cases potentially exposed to rabies could contribute to improving PEP prescribing practices to both reduce unnecessary costs associated with PEP and reach the target of zero human deaths due to rabies by 2023. METHODS: A cross-sectional survey of 50 clinicians' management of human cases potentially exposed to rabies was conducted in 13 health centers in high-rabies-risk areas of Bhutan during February-March 2016. RESULTS: Data were collected on clinicians' management of 273 human cases potentially exposed to rabies. The 50 clinicians comprised health assistants or clinical officers (55%) and medical doctors (45%) with a respective median of 19, 21 and 2 years' experience. There was poor agreement between clinicians' rabies risk assessment compared with an independent assessment for each case based on criteria in the National Rabies Management Guidelines (NRMG). Of the 194 cases for which clinicians recorded a rabies risk category, only 53% were correctly classified when compared with the NRMG. Clinicians were more likely to underestimate the risk of exposure to rabies and appeared to prescribe PEP independently of their risk classification.. Male health assistants performed the most accurate risk assessments while female health assistants performed the least accurate. Clinicians in Basic Health Units performed less accurate risk assessments compared with those in hospitals. CONCLUSIONS: This study highlights important discrepancies between clinicians' management of human cases potentially exposed to rabies and recommendations in the NRMG. In particular, clinicians were not accurately assessing rabies risk in potentially exposed cases and were not basing their PEP treatment on the basis of their risk assessment. This has significant implications for achieving the national goal of eliminating dog-mediated human rabies by 2030 and may result in unnecessary costs associated with PEP. Recommendations to improve clinician's management of human cases potentially exposed to rabies include: reviewing and updating the NRMG, providing clinicians with regular and appropriately targeted training about rabies risk assessment and PEP prescription, and regularly reviewing clinicians' practices.

Ambient Mass Spectrometry in Cancer Research.

Ambient ionization mass spectrometry was developed as a sample preparation-free alternative to traditional MS-based workflows. Desorption electrospray ionization (DESI)-MS methods were demonstrated to allow the direct analysis of a broad range of samples including unaltered biological tissue specimens. In contrast to this advantageous feature, nowadays DESI-MS is almost exclusively used for sample preparation intensive mass spectrometric imaging (MSI) in the area of cancer research. As an alternative to MALDI, DESI-MSI offers matrix deposition-free experiment with improved signal in the lower (<500m/z) range. DESI-MSI enables the spatial mapping of tumor metabolism and has been broadly demonstrated to offer an alternative to frozen section histology for intraoperative tissue identification and surgical margin assessment. Rapid evaporative ionization mass spectrometry (REIMS) was developed exclusively for the latter purpose by the direct combination of electrosurgical devices and mass spectrometry. In case of the REIMS technology, aerosol particles produced by electrosurgical dissection are subjected to MS analysis, providing spectral information on the structural lipid composition of tissues. REIMS technology was demonstrated to give real-time information on the histological nature of tissues being dissected, deeming it an ideal tool for intraoperative tissue identification including surgical margin control. More recently, the method has also been used for the rapid lipidomic phenotyping of cancer cell lines as it was demonstrated in case of the NCI-60 cell line collection.

Angiotensin converting enzyme in the human basal forebrain and midbrain visualized by in vitro autoradiography.

angiotensin converting enzyme converts angiotensin I to angiotensin II, a peptide that plays an important role in the central regulation of blood pressure and fluid and electrolyte homeostasis. However, the distribution of this enzyme in the human brain has not been well described. In this study, angiotensin converting enzyme was mapped in the human basal forebrain and midbrain by using quantitative in vitro autoradiography employing a derivative of a potent converting enzyme inhibitor, 125I-351A, as radioligand. This radioligand binds specifically and with high affinity to angiotensin converting enzyme and also exhibited these properties in binding to slide-mounted sections of human basal ganglia. In the basal ganglia, high levels of binding of 125I-351A are found in the caudate nucleus, putamen, nucleus accumbens, both divisions of the globus pallidus, and substantia nigra pars reticulata. High densities of labelling also occur in the ventral pallidum. In the hypothalamus, a moderate level occurs in the paraventricular and supraoptic nuclei, and a diffuse, low level of binding is found throughout the periventricular region. The organum vasculosum of the lamina terminalis, one of the circumventricular organs, displays the highest concentration of binding. The choroid plexus contains only moderate density of labelling in contrast to other mammalian species previously studied. Major fibre tracts are devoid of activity except for the posterior limb of the internal capsule, which contains fascicles of intense activity. In the midbrain, a moderate density of binding is detected in the periaqueductal gray. The dorsal, central linear, and, more caudally, the centralis superior medialis raphe nuclei also contain moderate densities of labelling. Angiotensin converting enzyme is heterogeneously distributed in the caudate nucleus and putamen, with distinct patches of high concentration surrounded by a matrix of diffuse, lower levels. In the caudate nucleus, these patches of high binding corresponded to striosomes since they register with acetylcholinesterase-poor zones. The high concentration of angiotensin converting enzyme found in the basal ganglia suggests that the enzyme may be involved in processing neuropeptides that occur in high concentrations in these structures. Possible substrates for converting enzyme include not only angiotensin I but also substance P and enkephalins, which are also concentrated in striosomes.

Intracellular recording of magnocellular preoptic neuron responses to olfactory brain.

The magnocellular preoptic nucleus of the rat supplies centrifugal input to the olfactory bulb as well as projecting to other olfactory-related areas. The extent to which the piriform and entorhinal cortices can influence the activity of magnocellular preoptic neurons and hence that of the olfactory bulb were examined using intracellular in vivo recording. Stable recordings were obtained in 58 neurons impaled in the magnocellular preoptic nucleus. Antidromic responses occurred on stimulating olfactory bulb (15), piriform cortex (14), or entorhinal area (eight). Monosynaptic excitation was evoked by piriform (27 of 37 tested) and entorhinal cortex (15 of 32 tested) stimulation with polysynaptic inhibition occurring in seven and five neurons, respectively. Polysynaptic as well as antidromic excitation by olfactory bulb stimulation occurred in four; a further 28 tested responded polysynaptically. No response to olfactory bulb stimulation was monosynaptic. In stable impalements, 29 neurons discharged spontaneously in the absence of applied current. Lucifer Yellow and Neurobiotin were used to label 16 cells. All but one had smooth dendrites with soma diameters ranging from 8 to 24 microm. These results provide a framework in which magnocellular preoptic neurons can influence olfactory processing by direct action on the olfactory bulb, which action can be boosted by positive feedback from the bulb through the olfactory piriform and entorhinal cortices.

Effects of lesions in the horizontal diagonal band nucleus on olfactory habituation in the rat.

The nucleus of the horizontal limb of the diagonal band, a component of the basal forebrain magnocellular complex affected in Alzheimer type dementia, supplies centrifugal innervation to the olfactory bulb. We have tested the hypothesis that horizontal limb of the diagonal band lesions will interfere with olfactory memory in a simple olfactory test paradigm. Lesions occupied a restricted volume, approximately 20%, of medial horizontal limb of the diagonal band. There was interference with habituation of investigation latency and duration, six and 16 days after lesioning. It is concluded that bilateral partial lesions of the medial nucleus of the horizontal limb of the diagonal band interfere with habituation memory for odours.

Evidence for bursting pacemaker neurones in cultured spinal cord cells.

Intracellular recordings were made from dissociated mouse spinal cord cells in primary culture. One type of spinal cord neurone, with a large cell body (40-50 micron), 3-5 short neurites, and a mean resting potential of -65 mV, was found to fire rhythmic bursts of action potentials with a phase duration of approximately 1s when the membrane potential was depolarized to -55 mV. These bursts did not arise from spontaneous synaptic input, but appeared to result from endogenous ionic conductance properties of the membrane resembling those observed in molluscan bursting pacemaker neurones. Ionic conductances underlying this bursting activity were studied pharmacologically by local application of ionic conductance blockers. Pacemaker potentials depended on Na+ conductance, since tetrodotoxin and Na-free medium were the most potent agents for blocking spontaneous rhythmic activity. However, a Ca2+ conductance was involved in the depolarizing phase of membrane potential oscillations, since Ba2+ application increased oscillation amplitude. Action potentials observed during the bursts were Na+- and Ca2+-dependent. They did not differ significantly from those observed in other spinal cord neurones in culture. Application of tetraethylammonium, CoCl2, BaCl2 and 4-aminopyridine revealed at least three different potassium conductances which controlled this bursting pacemaker activity. A delayed potassium conductance controlled spike duration, a Ca-dependent potassium conductance controlled the duration of the burst and underlay the hyperpolarizing phase terminating the burst, and finally, a transient potassium conductance appeared to be involved in the control of phase duration. The demonstration that spinal cord neurones growing in monolayer culture display typical bursting pacemaker activity raises the possibility that bursting pacemaker neurones in the mammalian spinal cord may be involved in a phasic pattern generator that could control such activities as walking and the respiratory rhythm.

Intracellular responses of olfactory bulb granule cells to stimulating the horizontal diagonal band nucleus.

The effects of centrifugal afferents on membrane potentials of identified granule cell layer using evoked field potential profiles, and trans-synaptic activation via antidromic stimulation of output cell axon collaterals. Intracellular recordings maintained for 4-30 min showed complex spontaneous spike discharges and allowed characterization of the cell's input resistance, and on some occasions its morphology following intracellular injection of Lucifer Yellow. Stimulation in the nucleus of the horizontal limb of the diagonal band, but not surrounding regions, produced hyperpolarizing responses in 13 of 27 cells in the granule cell layer; four of these were morphologically identified as granule cells of two types, in five the responses had reversal potentials more negative than the resting potential, and six were identified as granule cells by monosynaptic activation from output axon collaterals. A different set of three cells in the granule cell layer responded with depolarization. The results are consistent with the inhibition of tonic activity of granule cells by the nucleus of the horizontal limb of the diagonal band, leading to disinhibition of mitral and tufted cells via dendrodendritic synapses of granule cells on mitral/tufted cell secondary dendrites.

Effect of stimulating the nucleus of the horizontal limb of the diagonal band on single unit activity in the olfactory bulb.

The effects of centrifugal afferents on single unit discharge in the main olfactory bulb were studied in anaesthetized rats. Recording with extracellular micropipettes revealed spontaneous firing in all bulb layers. Units were located to different laminae using evoked field-potential profiles and histological verification. Output neurons were identified by antidromic response to stimulation of the lateral olfactory tract. Single- or brief multiple-pulse stimulation in the nucleus of the horizontal limb of the diagonal band, but not in adjacent regions, facilitated 17 out of 27 mitral cells with no effect on 10, but inhibited 21 out of 33 granule cell layer units with no effect on 12. Of 13 presumed tufted cells, six were facilitated and the rest unaffected. In contrast, stimulation of olfactory cortex inhibited mitral cells and facilitated most granule layer cells. The results are consistent with an inhibition of tonic granule cell discharge by the horizontal diagonal band nucleus, with resultant disinhibition of mitral cells via the dendrodendritic synapses of granule cells on mitral cell secondary dendrites.

Localization of binding sites for calcitonin gene-related peptide in rat brain by in vitro autoradiography.

The distribution of binding sites for calcitonin gene-related peptide (CGRP) in rat brain were studied using in vitro autoradiography. In a radioreceptor assay using [125I]human calcitonin gene-related peptide as the radioligand, with cerebellar cortical membranes, rat calcitonin gene-related peptide had a binding affinity constant of 1.16 +/- 0.23 X 10(10) M-1 and a site concentration of 43.4 +/- 3.4 fmol/mg protein. In this system, human calcitonin gene-related peptide had a binding affinity constant of 3.9 +/- 0.7 X 10(9) M-1 whereas salmon calcitonin was very weak with a binding affinity constant of only 6.8 +/- 4.0 X 10(5) M-1. CGRP binding localized by in vitro autoradiography, using [125I]rat calcitonin gene-related peptide, had a characteristic distinct distribution in the rat brain. There were high concentrations of binding found over the accumbens nucleus, the organum vasculosum of the lamina terminalis, ventral caudate putamen, median eminence, the arcuate nucleus, lateral amygdaloid nucleus and lateral mammillary nucleus, the superior and inferior colliculi, pontine nuclei, molecular and Purkinje cell layers of the cerebellar cortex, the nucleus of the solitary tract, the inferior olivary nuclei, hypoglossal complex and the vestibular and cochlear nuclei. The distribution of these binding sites suggests multiple roles for CGRP in the central nervous system including auditory, visual, gustatory and somatosensory processing, and in neuroendocrine control.

Evidence for a new subclass of calcitonin/ calcitonin gene-related peptide binding site in rat brain.

Binding sites for calcitonin and calcitonin gene-related peptide are widely distributed in the central nervous system. In this study, binding of [(125)I]-alpha-rat calcitonin gene-related peptide and [(125)I]-salmon calcitonin in adjacent sections of rat brain revealed clearly distinct patterns of binding in most regions although in some restricted areas such as parts of the ventral striatum, including the nucleus accumbens, there was some overlap in the patterns of binding. In the primary olfactory cortex, which bound only calcitonin gene-related peptide, salmon calcitonin was very weak in inhibiting the binding of calcitonin gene-related peptide. In the nucleus accumbens, high affinity binding of calcitonin and calcitonin gene-related peptide at their homologous receptors was observed, with affinity constants for calcitonin and calcitonin gene-related peptide of 1.4 x 10(9) M(?1) and 1.2 x 10(9) M(?1) respectively. Cross competition studies in this nucleus demonstrated that salmon calcitonin was able to compete for [(125)I]-rat calcitonin gene-related peptide labelled sites with high affinity, with an affinity constant of 0.8 x 10(9) M(?1). However, rat calcitonin gene-related peptide was less potent in inhibiting the binding of [(125)I]-salmon calcitonin labelled sites with only 28% inhibition at 10(?6)M. Further characterization of the calcitonin sensitive calcitonin gene-related peptide labelled sites demonstrated that a range of calcitonin analogs inhibited the binding of [(125)I]-rat calcitonin gene-related peptide with the same order of potency as the analogs competed for [(125)I]-salmon calcitonin labelled sites. Digital substraction mapping revealed calcitonin-sensitive calcitonin gene-related peptide binding sites over parts of the ventral striatum, including mid-caudal nucleus accumbens and fundus striati; over the lateral border of the lateral bed nucleus of the stria terminalis; part of the central amygdaloid nucleus; the organum vasculosum of the lamina terminalis and area postrema and over the wings of the dorsal raphe. These results demonstrate the existence of a new subtype of calcitonin/calcitonin gene-related peptide binding site, which has high affinity for the two otherwise biochemically distinct peptides.

Effects of inactivation of the magnocellular preoptic nucleus of olfactory bulb processing.

The magnocellular nucleus (MCPO) was inactivated in anaesthetized rats, using muscimol, a gamma-amino butyric acid ergic agonist, in order to examine the effect of suppression of its tonic activity on extracellular unit firing in the granular (GRL), mitral (MCL) and external plexiform (EPL) layers of the olfactory bulb (OB). In GRL there was a significant increase in unit activity during the first hour after muscimol injection (30 ng), followed by a significant decrease in activity during the following hour. No effect on activity in MCL was seen after muscimol injection into the MCPO. Unit activity in EPL increased during the second hour post-injection. It was concluded that MCPO plays an important part in regulating the balance between granule and tufted cell activity.

Comparison of uterine activity induced by nipple stimulation and oxytocin.

Intermittent nipple stimulation has been proposed as a substitute for exogenous oxytocin infusion in the performance of contraction stress tests. To compare the uterine activity produced by these two methods, we studied a group of 45 term pregnant women undergoing indicated inductions of labor. Twenty-five patients had nipple stimulation and 20 patients received oxytocin infusions according to a study protocol. The two groups were similar in all obstetric parameters. Pre- and posttest uterine activity was measured by internal tocodynamometry and quantified in Montevideo units. A significant increase in uterine activity occurred in both groups (P less than .01). Regular uterine activity (three contractions in ten minutes) was achieved more rapidly (P less than .005), but at a lower level (P less than .001) in the nipple stimulation group. Pre- and posttest tonus did not change significantly in either group. In the nipple stimulation group, five patients (20%) did not achieve adequate contraction patterns after 15 stimulation-rest cycles (a total of 110 minutes) and three subjects (12%) experienced uterine hyperstimulation. These observations suggest that exogenous oxytocin and intermittent nipple stimulation may not have equivalent effects on uterine contractility. Therefore, it may not be justified to substitute one technique for the other or to use the same criteria for interpretation of contraction stress tests produced by both techniques.

Lesions in the magnocellular preoptic nucleus decrease olfactory investigation in rats.

The nuclear complex of the horizontal limb of the diagonal band and the magnocellular preoptic nucleus, components of the basal forebrain magnocellular system affected in Alzheimer-type dementia, supply centrifugal innervation to the olfactory bulb. The lateral magnocellular preoptic nucleus provides significant GABAergic input. Since its stimulation may facilitate olfactory bulb mitral cells, we have investigated the effect of sub-total electrolytic lesions in this nucleus on performance in a simple test of olfactory investigation and its habituation. Two groups of rats used with lesions which occupied restricted volumes, approximately 30 and 15% of the magnocellular preoptic nucleus. Behaviorally, there was interference with olfactory investigation, with increased investigation latency and decreased investigation times, the group with larger lesions at 6 and 16 days after operation. There was no significant effect of the smaller lesions. No effects on patterns of olfactory habituation or discrimination were seen. The impairment of olfactory investigation could not be explained by interruption of medial forebrain bundle fibres traversing the nucleus. It is suggested that bilateral partial destruction of magnocellular preoptic neurones may produce significant deficits in either olfactory sensitivity or olfactory motivation.

Intrastriatal dopaminergic agents, muscarinic stimulation, and GABA antagonism compared for rotation responses in rats.

We tested the common hypothesis that rotation on systemic injection of dopaminergic agents in rats with unilateral 6-hydroxydopamine lesions of the substantia nigra is attributable to unequal stimulation of dopamine receptors between the two striata. No rotation occurred when dopamine, apomorphine or amphetamine were injected into dorsal striatum or nucleus accumbens of intact, unanesthetized rats. Intrastriatal haloperidol elicited ipsiversive postural deviation only in conjunction with hypermotility induced by systemic amphetamine. In unilateral 6-hydroxydopamine-lesioned rats, intrastriatal apomorphine elicited rotation directed away from the side of its injection, whether intact or lesioned. Carbachol elicited short-latency rotation, contraversive to injection in dorsal striatum or nucleus accumbens, in both intact and 6-hydroxydopamine-lesioned rats. The rotation response to carbachol was suppressed by atropine administered systemically or into the site of intrastriatal carbachol. Picrotoxin or bicuculline produced contraversive rotation or contralateral myoclonic jerks on injection into the striatum in intact rats. The results show that asymmetric stimulation of striatal dopamine receptors is not sufficient to cause rotation, unless the receptors have been denervated. On the other hand, asymmetric stimulation muscarinic receptors is in itself enough to produce the imbalance of gamma-aminobutyric acid (GABA)ergic striatal outputs responsible for rotation.

Olfactory bulb output neurons excited from a basal forebrain magnocellular nucleus.

We present intracellular data which demonstrates a unique facilitatory centrifugal influence on the output cells of the olfactory bulb; the source being the lateral component of the nucleus of the horizontal limb of the diagonal band (HDB), part of the basal forebrain magnocellular complex. Damage to this facilitatory HDB influence may explain the loss of olfactory sensitivity seen early in Alzheimer's disease in which pathological changes occur in the basal forebrain.

Catecholamine uptake sites in mouse brain: distribution determined by quantitative [3H]mazindol autoradiography.

Because of the importance of the mouse brain catecholamine system in the study of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and because little information is available concerning the chemical neuroanatomy of the mouse, catecholamine uptake sites were mapped in C57 black mouse brain using [3H]mazindol autoradiography. Displacement studies with known dopamine (DA) and noradrenaline (NA) uptake blockers showed that binding in the striatum was entirely to DA uptake sites, while binding in the locus coeruleus was to NA uptake sites only. By using the selective noradrenergic uptake blocker desmethylimipramine (DMI), a complete map of both DA and NA uptake sites was generated. The mesostriatal DA system was the most clearly labelled and uptake sites were seen better in striatal terminals than the substantia nigra. Within the noradrenergic system, highest binding levels were seen over the locus coeruleus, although it was unclear whether these uptake sites were on cell bodies or terminals from the lateral tegmental noradrenergic system. These maps of the catecholamine uptake system in mouse brain provide a baseline for study of newly discovered neurotoxins and ageing processes.

Bilateral projection of nigro-collicular neurons: An electrophysiological analysis in the rat.

In response to stimulation of the deep layers of the superior colliculus (SC) in the rat, nigral neurons were antidromically activated (latency 0.5-1.5 msec). Effective stimulations were located in ipsi- or contralateral SC deep layers lying above the central gray (CG) or more laterally; nigro-collicular neurons are exclusively located in the pars reticulata of the substantia nigra (SN) and some of them project both to ipsi- and contralateral SC. The functional significance of this nigral projection is discussed.

Regional and temporal effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine on dopamine uptake sites in mouse brain.

When the regional effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) on brain dopamine uptake sites in C57 Black mice were studied using [3H]mazindol autoradiography, marked regional differences in effect were seen: the mesolimbic system was less affected than the nigrostriatal tract and within each system the effect was more severe in the terminal fields of the striatum than in the cells of origin. Within the striatum itself there was inhomogeneity of effect, with relative sparing of the dorsomedial aspect compared to the remainder. Complete recovery of [3H]mazindol binding to striatal membranes occurred over 12 months, while dopamine levels recovered more slowly. This supports the concept that MPTP has a highly selective effect within dopaminergic systems and that the initial effect is more pronounced on distal terminals compared to cell bodies. The possibility that recovery of mazindol binding with time may be associated with terminal regrowth needs to be investigated further.

Unit responses of intralaminar thalamus to midbrain and medullary stimulation and effects of conditioning caudate and hippocampal stimuli.

Single units responding to heterotopic somatic stimuli, on extracellular recording in thalamic intralaminar and neighbouring nuclei, also responding to stimulation of the midbrain tegmentum or the medullary magnocellular reticular formation. Consideration of response latencies suggested that some monosynaptic projections from both midbrain and medulla may be received in nuclei centralis lateralis, centrum medianum-parafascicularis complex, and medial ventralis lateralis. Responses to brainstem of nuclei medialis dorsalis, lateralis posterior were of considerably longer latency. There was no correlation between shortness of latency and following-rate of unit responses; the ability of intralaminar neurons to follow rapidly-repeated brainstem stimuli is inferred to be limited by inhibitory processes rather than by synaptic interruptions in the afferent pathway. Conditioning stimuli to caudate nucleus or hippocampus suppressed most intralaminar responses to midbrain stimuli, the shortest-latency responses included, suggesting that inhibitory effects could be exerted at the thalamic level, perhaps directly on the responsive neurone.

Multiunit bursts in rat pallidum during grooming and stereotyped jaw movements.

Extracellular recordings from the globus pallidus of awake, unrestrained rats showed a distinctive bursting activity during grooming behaviour and in periods of stereotyped jaw movements induced by amphetamine (3 or 5 mg/kg IP) or apomorphine (2 mg/kg SC). During stereotyped licking, there was one burst for each outward movement of the tongue. The bursts were shown to consist of several separate unit spikes firing so as to produce a fusiform envelope of amplitudes, suggesting an ordered recruitment of pallidal neurons related to licking.