Determination of amphetamine and methamphetamine in human hair by headspace solid-phase microextraction and gas chromatography with nitrogen-phosphorus detection.

A simple and rapid method for the determination of amphetamine (AP) and methamphetamine (MA) in human hair was developed by headspace solid-phase microextraction (SPME) and gas chromatography with nitrogen-phosphorus detection (GC-NPD). The hair (1 mg) was dissolved in 0.2 ml of a 5 M sodium hydroxide solution in a tightly sealed vial by shaking at 75 degrees C for about 5 min. In order to adsorb AP and MA on the SPME fiber, 100 microm of polydimethylsiloxane fiber was exposed to the headspace of the vial, and the vial was heated at 55 degrees C for 20 min. Then the fiber was removed from the vial and inserted into the injection port of the GC-NPD system using a CBJ-17 capillary column. The compounds adsorbed on the fiber were analyzed by exposing the fiber at 220 degrees C for 30 s in the GC injection port. By using this method, AP and MA in human hair could be analyzed simply and rapidly without any interference from coexisting substances. The percentages of AP and MA extracted from human hair by the SPME method were 48 and 62%, respectively, and relative standard deviations were below 10% (n=5). The calibration curves for AP and MA were linear in the ranges of 0.4-15 and 4-160 ng/mg hair, respectively. The detection limits of AP and MA at a signal-to-noise ratio of three were 0.1 and 0.4 ng/mg hair, respectively. This method could be applied to the analysis of an abuser's hair sample.

Spatiotemporal appearance of developing LHRH neurons in the rat brain.

To obtain insight into the development of the heterogeneous intracerebral populations of luteinizing hormone-releasing hormone (LHRH) neurons, their spatiotemporal appearance was examined at different stages in normal rat embryos, in nasal epithelial explants in vitro, and in intrauterine nasal-operated embryos. Following the appearance of nerve cell adhesion molecule in the nasal placode at embryonic day (E) 12.5, LHRH neurons, generated in the nasal placode at E13.5, penetrated the forebrain vesicle (FV) by E14.5-15.5. After E16.5, as the FV elongated to form the olfactory bulb, the migrating neurons traversed posteriorly through the interhemispheric space to penetrate the septopreoptic (S-P) area. By E18.5, LHRH neurons were detected in the preoptic-diagonal band (P-D) area as well as in the S-P region, along with some scattered extrahypothalamic LHRH neurons. To determine the source of these neurons, we separately cultured dissected parts of E12.5 nasal pit epithelium. Neuronal generation was predominantly from the medial wall epithelium (NAP), but some LHRH neurons originated in the roof epithelium. Cocultures of the NAP (E12.5) with the FV, median eminence-arcuate complex, Rathke's pouch, mesencephalon, or medulla oblongata from E14.5 embryos revealed the ability of LHRH cells to penetrate all of these tissues. Uni- or bilateral nasal destruction was conducted at E16.5 or E15.5, respectively, and examined at E18.5 and E21.5. In the operated embryos, most LHRH neurons were present in the P-D system and some in the S-P area. This finding suggests that the neurons generated before E15.5 are primarily predisposed to form the P-D system, whereas those derived afterward form the S-P system.

Destruction of olfactory inputs affects the morphogenesis of the telencephalon in rats.

We unilaterally destroyed the nasal radix of rat embryos on day 15.5 of gestation (E15.5) in utero so as to block the olfactory inputs to the ipsilateral forebrain vesicle. The embryonic brains were examined after 6 days' survival (E21.5). In the deafferented half of the brain, LHRH neurons were significantly reduced in number, indicating the successful blocking of the olfactory input. On the deafferented side, the olfactory bulb failed to develop, and the telencephalic hemisphere, small in size, accompanied various histogenetic retardations in the primary olfactory cortex, in the cortical plate, and in the hippocampal formation. The striatum revealed remarkable structural differences between the ipsilateral and contralateral sides: on the ipsilateral side, the striatum was small in size and displayed numerical reductions of immunoreactive tyrosine hydroxylase (TH) fibers and substance P (SP) neurons in comparison with those in the contralateral one; in the substantia nigra, TH neurons and SP fibers were less numerous on the deafferented side. There were no remarkable differences in the distribution of TH neurons in the hypothalamus. In view of these sequential histogenetic alterations, it can be assumed that the olfactory inputs play a key role in the telencephalic morphogenesis.

Strategy for health and safety management at an automobile company--from the prevention of low back pain to Toyota's Verification of Assembly Line (TVAL).

This paper reports on a strategy to improve and renovate assembly lines, including countermeasures to prevent low back pain during the past two decades at Toyota Motor Co. Since 1975, there have been problems with low back pain at Toyota's vehicle assembly lines. To deal with these low back pain problems, it was necessary to determine their causes and to quantitatively evaluate the burden on workers. For this purpose, functional burden indexes were developed, that is, a posture burden point and a weight burden point were determined to assess the load on the low back, and a low extremity point and a squatting posture point were determined to assess the burden on the leg. The functional burden index, however, could be applied only to specific human functions, not to human functions in general. Since there are about 400 kinds of working patterns in vehicle assembly lines, comprehensive burden index was required to estimate overall burden of such work. Thus, we developed Toyota's Verification of Assembly Line (TVAL), an index for assessing the physiological stress of an assembly line work, in which an equivalent bicycle ergometer workload is calculated from electromyograms taken of 20 different muscles under actual working conditions. At present, TVAL is used to measure physiological burden of assembly work in order to give priority to improvements, and to objectively demonstrate the effects of such improvements at Toyota.

In vitro analysis of the centripetal migration mechanisms of developing LHRH neurons.

This study was designed to gain insight into the underlying mechanisms of the centripetal migration of developing LHRH neurons. The medial wall of the nasal pit (NAP) of 12.5-day-old rat embryos (E12.5) was cultured singly or together with the E12.5 medial-basal wall of the forebrain vesicles (mFV) or with the E14.5 median eminence-arcuate complex (ME-Arc). Further, the NAP was cultured with the mFV and ME-Arc or with the mFV and nasal mesenchyme (NM), which lay between the mFV and the NAP, of E12.5 embryos (triple culture). The NAP gave rise first to fibers labeled with anti-neural cell adhesion molecules (NCAM) and then to LHRH neurons. In co-cultures, NAP- and brain-derived NCAM fibers connected the NAP and brain cultures, and frequently linked with each other to form knots at the periphery. LHRH neurons migrating along the NAP-derived fibers directly or indirectly entered brain cultures. In the latter case, the cells strayed along the way from the NAP-derived fibers to the brain-derived fibers at the knots and migrated retrogradely along the latter fibers to enter into the brain tissues; this occurred most frequently into the E14.5 ME-Arc. In triple cultures, abundant NCAM fibers emerging from the NAP were only found when the NM lay between the NAP and mFV; the fibers converged further to the mFV. These findings help elucidate the mechanisms underlying the centripetal LHRH cell migration from the NAP to the hypothalamus.

How the developing septo-preoptic medical basal hypothalamus stimulates the development of placode-derived LHRH neurons.

We examined the effects of the developing cerebral cortex (CC) and septo-preoptic medial basal hypothalamus (S-MBH) on the development of LHRH neurons in vitro. The serum-free basal culture medium (BCM) was supplemented with CC or S-MBH extracts prepared from 18.5-day-old embryos or from 2-day-old newborns, and the olfactory placode (NAP) of 12-day-old embryos was cultured. The migration of LHRH neurons was found on Day 3 in the cultures supplemented with the embryonic S-MBH extract (Group 3), where the cell development proceeded showing a numerical increase of the cells and the elongation of neurites. In cultures supplemented with the newborn S-MBH extract (Group 5), the cell development was less intensive in comparison with that of Group 3, while in cultures which had no brain extracts (Group 1), the neurons failed to survive a long term culture. The effects of the CC were less than of S-MBH extracts. Analysis of the protein composition of the extracts by electrophoretic and immunoblotting examinations demonstrated a protein spot of 70-kD in the embryonic S-MBH extract. Because the protein spot was identified to be alpha-fetoprotein (AFP), we further examined the effects of AFP. When the anti-AFP immunoglobulin was added to the Group 3 culture, the stimulative effects of the embryonal extract were inhibited, and the addition of AFP to Group 1 cultures did not show stimulative effects. We conclude that the developing S-MBH, the migrating target of LHRH neurons, contains some essential factors for the development of LHRH neurons, but further analysis is needed to determine the chemical natures of these factors.

In vitro development of placode-derived LHRH neurons: possible involvement of alpha-fetoprotein.

Using the olfactory placode (NAP) of 12.5-day-old and vomeronasal organ (VNO) of 14.5-day-old (E12.5, E14.5) rat embryos, we examined in vitro the roles of brain tissues in the development of LHRH neurons. The culture was performed singly or in combination with various brain tissues of E12.5 and E14.5 embryos. Furthermore, a serum-free basal culture medium was supplemented with a water extract of cerebral cortex or septopreoptic-medial basal hypothalamus (S-MBH) of E18.5 embryos and 2-day-old (N2) newborns. In cocultures, many LHRH cells derived from the NAP or VNO migrated into the collocated tissues of the forebrain vesicles and medial basal hypothalamus, especially into the latter, along neural cell adhesion molecule (NCAM)-positive fibers projecting from the NAP and/or the brain tissues, especially in the case of the medial basal hypothalamus. The effects of the brain extracts were evaluated by differentiation, migration, neurite extension, and survival of LHRH neurons. E18.5 S-MBH extract was most effective. In the analysis of the protein composition of the extracts, alpha-fetoprotein (AFP) was determined only in the E18.5 S-MBH extract. When anti-AFP IgG was added to the cultures containing E18.5 S-MBH extract, the stimulative effects of the extract were reduced to the level of the N2 S-MBH extract. When we cultured the NAP by adding AFP in the basal culture medium, the development of LHRH cells was somewhat promoted. The actual roles of AFP thus remain to be further elucidated.

Migration of LHRH neurons derived from the olfactory placode in rats.

Using the olfactory placode of 12.5- and 14.5-day-old (E12.5, E14.5) rat embryos, we examined the migration of LHRH neurons by in vivo intraventricular transplantation and in vitro organotypic culture systems. In the transplantation, the olfactory placode of E12.5 embryos was co-transplanted with the cerebral cortex and also with medial basal hypothalamus (MBH). LHRH neurons that had migrated into the co-transplanted brain tissues were fusiform, but those that had moved into the neuro-mesenchymal tissue were polyhedral. The migration occurred most conspicuously in the MBH. In our in vitro studies, we used E14.5 embryos; their vomeronasal organ was cultured with MBH, the olfactory cortex, and the septum of the telencephalon in two systems (piled-culture with an intervening transferrable membrane and co-culture). Among these brain tissues, the MBH was the most effective in inducing the development and migration of LHRH neurons. We further found synaptic junctions of immunonegative nerve fibers on immunoreactive LHRH neurons located in the septum of E16.5 and 17.5 embryos. These findings suggest that the MBH may lead the intraseptal migration of LHRH neurons by yielding certain substances after introducing the neurons into the medial aspect of forebrain vesicles. The early development of the neuronal connection may further promote the migration of LHRH neurons.

Temporal bone histopathology in trisomy 18 syndrome: a report of two cases.

Temporal bone histopathological findings of two patients with trisomy 18 syndrome are described. Many of the abnormalities previously described were seen in the present cases; namely, atresia of the external auditory canal, aberrant course of the tensor tympani muscle, malformed stapes, aberrant course of the facial nerve with an obtuse angulation at the first genu and displacement of geniculate ganglion cells into the internal auditory canal, shortened cochlea with decreased spiral ganglion cell population, and vestibular anomalies, such as bony and membranous blockage of the superior semicircular canal. Moreover, an extremely underdeveloped malleus and incus continuous with a persistent Meckel's cartilage were observed.

Anomalies of the auditory organ in Potter's syndrome. Histopathological findings in the temporal bone.

Histopathological findings in the temporal bone are described in a newborn infant, diagnosed as having Potter's syndrome. The infant has severely malformed low-set ears bilaterally and a small lower jaw; autopsy findings showed bilateral renal agenesis and pulmonary hypoplasia. The temporal bone indicated the deformities of the inner ear, classified as Mondini-type, complicated by extensive deformities to the external ear and middle ear, including absence of auditory ossicles, atresia of the oval window, abnormal course of the facial nerve, and hypoplastic external auditory canal. The cochlear membranous labyrinth showed nearly normal form in the upper turn, but severe hypoplasia in the basal turn, which was an unusual cochlear anomaly.