Objective. To assess the face, content, and construct validity of the Voxel-Man TempoSurg Virtual Reality simulator. Participants and Methods. 74 ear, nose, and throat (ENT) surgeons participated. They were assigned to one of two groups according to their level of expertise: the expert group (n = 16) and the novice group (n = 58). The participants performed four temporal bone dissection tasks on the simulator. Performances were assessed by a global score and then compared to assess the construct validity of the simulator. Finally, the expert group assessed the face and content validity by means of a five-point Likert-type scale. Results. experienced surgeons performed better (p < .01) and faster (p < .001) than the novices. However, the groups did not differ in terms of bone volume removed (p = .11) or number of injuries (p = .37). 93.7% of experienced surgeons stated they would recommend this simulator for anatomical learning. Most (87.5%) also thought that it could be integrated into surgical training. Conclusion. The Voxel-Man TempoSurg Virtual Reality simulator constitutes an interesting complementary tool to traditional teaching methods for training in otologic surgery.

Telemedicine is emerging as a reliable tool for examining and delivering treatment to patients during the acute phase of stroke but little is known about patient outcome in this setting. We compared time to examination and outcomes between two groups of consecutive patients treated with intravenous of recombinant tissue plasminogen activator (rt-PA) at the acute phase of ischemic stroke between 1st July 2013 and 30th June 2014. One group of patients was managed via a telestroke system (Virtuall, established in Lorraine, east France) and the second at the bedside in the stroke unit of Nancy Hospital. During this period, 27 patients received rt-PA via the telestroke system and 70 in the stroke unit. Both groups presented similar mean age and severity of stroke. There was no significant difference in median onset to needle times (180 minutes vs 170 minutes, P = 0.35), despite a longer door to needle time in the telestroke system group (86 minutes vs 55 minutes, P < 0.001). Outcome was similar in both groups: intracranial haemorrhage (19% vs 24%, P = 0.60); death (30% vs 20%, P = 0.42); and patient independence at 3 months (modified Rankin Scale score ≤ 1: 30% vs 31%, P = 1.00). This study demonstrates that the telestroke system, Virtuall, is as safe and effective in initiating rt-PA treatment as bedside evaluation in an experienced stroke unit, despite a longer intra-hospital time. Le devenir des patients victimes d’accidents vasculaires cérébraux (AVC) évalués et traités par télémédecine reste peu connu. Nous avons comparé de façon rétrospective le devenir de deux groupes de patients, traités pour un AVC ischémique, par activateur du plasminogène tissulaire recombinant (rt-PA) intraveineux, inclus de façon consécutive entre le 1er juillet 2013 et le 30 juin 2014. Le premier groupe a été examiné et traité par l’intermédiaire d’un système de téléAVC développé en région Lorraine, dans le Nord-Est de la France, appelé « Virtuall », et le second directement au sein de l’unité neurovasculaire (UNV) expérimentée du centre hospitalier universitaire de Nancy. Durant cette période, 27 patients ont été traités par rt-PA via téléAVC et 70 directement au sein de l’UNV. Les deux groupes, téléAVC et UNV, avaient les mêmes caractéristiques cliniques d’âge et de degré de sévérité. Les délais médians séparant la survenue de l’AVC et l’instauration du traitement n’étaient pas significativement différents (180 minutes vs 170 minutes, p = 0,35), malgré un délai entre l’admission et le traitement plus élevé dans le groupe évalué par télémédecine (86 minutes vs 55 minutes, p < 0,001). Le devenir des patients fut similaire avec des pourcentages d’hémorragies cérébrales (19 % vs 24 %, p = 0,60), de décès (30 % vs 20 %, p = 0,42) et de patients autonomes à 3 mois (score modifié de Rankin ≤ 1, 30 % vs 31 %, p = 1,00) comparables entre les deux groupes. Nos résultats démontrent que notre système de télémédecine, Virtuall, est un moyen aussi sûr et efficace d’examen et de prise de décision de traitement dans ce contexte que l’évaluation directe au sein d’une UNV, en gardant à l’esprit un temps intra-hospitalier accru.

PURPOSE:To define the activity and toxicity of preoperative chemotherapy and postoperative concomitant chemoradiotherapy in patients with carcinoma of the esophagus, and to determine the effect on survival in patients treated with this approach.PATIENTS AND METHODS:Patients were treated with two 21-day cycles of induction chemotherapy with cisplatin 100 mg/m2 on day 1, 5-fluorouracil (5-FU) 800 mg/m2/day continuous infusion on days 1-5, and leucovorin 100 mg/m2 every four hours on days 1-5. Surgical resection was performed if feasible (and could also be performed prior to chemotherapy). Patients then received radiotherapy (50 to 60 Gy) every other week x five to six weeks, concomitantly with 5-FU 800 mg/m2 continuous infusion daily and hydroxyurea 1 g twice daily x five days.RESULTS:Forty-six patients were treated. With a minimum follow-up of 58 months, the median survival for the entire group was 16 months; the median survivals for patients with squamous carcinoma and adenocarcinoma were 29 months and 12 months, respectively. Toxicities of induction chemotherapy were severe neutropenia and mucositis; there was one toxic death. Toxicities of concomitant chemoradiotherapy were neutropenia, mucositis and esophagitis. There were five cases of radiation pneumonitis, one fatal.CONCLUSION:Induction chemotherapy and postoperative concomitant chemoradiotherapy can be added to surgical resection for carcinoma of the esophagus. Combined modality therapy, as reported here, produces long-term survival benefit, particularly in patients with squamous carcinoma. However, similar outcome results have been reported with less toxic and shorter treatment regimens as tested in randomized studies.

1. The rotation-mediated three-dimensional reaggregate culture system is uniquely suited for studies on developmental neurotoxicity. In this system, it is possible to reconstruct central neuronal pathways and follow their development. 2. Exposure to drugs of abuse including methamphetamine and methylenedioxyamphetamine or the appetite suppressant, fenfluramine, reduces monoamines in the cultures in a dose-dependent manner and interrupts normal monoaminergic development. 3. While the monoaminergic neurones may attain normal rates of development following drug removal, the affected neurones are not capable of overcoming the drug-induced insults and a deficiency in monoamines persists throughout development. 4. In addition, the production of immortalized monoclonal hybrid cells obtained by fusion of fetal mesencephalic neurones with a neuroblastoma has yielded cell lines expressing a dopaminergic phenotype. 5. Such cells have been useful in establishing the relationship of neurotoxicity to cell lineage and can serve as models for the study of the cellular and molecular mechanisms of neurotoxicity.

Three-dimensional, rotation-mediated reaggregate tissue cultures composed of rostral mesencephalic cells and corpus striatal cells were used to examine the short-term and persistent effects of methamphetamine on developing monoamine-containing neurons. Reaggregates were exposed to drug for one week. Reductions in reaggregate endogenous dopamine and serotonin levels occurred following treatment with methamphetamine during days 15-22 of culture over the concentration range 10(-7) to 10(-4) M. The highest methamphetamine concentration reduced dopamine and serotonin levels to 29 and 33%, respectively, of control values. Monoamine levels were reduced from control values after 3 days of exposure to 10(-4) M methamphetamine. No further reduction resulted from 4 additional days of drug treatment. In order to determine whether monoaminergic neurons would recover from the drug-induced deficit, reaggregates were exposed to 10(-4) M methamphetamine for 7 days and then grown in drug-free media for an additional 20 days. During the 20 day recovery period, monoamine levels in the control group increased with time in culture. After an initial rapid increase (recovery days 0-9), the level of monoamines in the recovery group remained at a constant proportion to the level in the control group suggesting that the monoaminergic neurons return to a rate of development similar to that seen in untreated cultures. However, this rate was not sufficient to overcome the reduction in monoamine levels produced by 7 days of methamphetamine treatment. The results indicate that the effects of methamphetamine on developing monoaminergic neurons are marked and persistent.

Developing central dopamine (DA)-containing neurons in rotation-mediated reaggregate tissue culture exposed to 10(-4) M methamphetamine for 1 week (at a time corresponding to the 2nd week of postnatal development) exhibit decreased DA levels DA neurons were visualized either by a histofluorescent method after 'loading' with exogenous DA, or by tyrosine hydroxylase (TH) immunocytochemistry. An apparent loss of histofluorescent DA cell bodies was observed due to a methamphetamine-induced reduction in exogenous DA accumulation. No decrease in the number of TH-immunoreactive neurons was observed. Thus, developing DA neurons when visualized by TH immunocytochemistry survive despite methamphetamine-induced reductions in DA levels.

To facilitate the study of trophic interactions between mesencephalic dopaminergic neurons and their target cells, clonal hybrid cell lines have been developed from rostral mesencephalic tegmentum (RMT) of the 14-day-old embryonic mouse employing somatic cell fusion techniques. Among the hybrid cell lines obtained, one contains a high level of dopamine (DA), another predominantly 3,4-dihydroxyphenyl-alanine (DOPA), and a third no detectable catecholamines. The hybrid nature of the cell lines is supported by karyotype analysis and by the expression of adhesion molecules as assessed by aggregation in rotation-mediated cell culture. The DA cell line shows neuronal properties including catecholamine-specific histofluorescence, neurite formation with immunoreactivity to neurofilament proteins, and large voltage-sensitive sodium currents with the generation of action potentials. In contrast to the pheochromocytoma cell line (PC12), the dopamine content of the DA hybrid cell line is depleted by low concentrations of N-methyl-4-phenylpyridinium ion (MPP+), the active metabolite of the neurotoxin N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP).

The purpose of the present study was to examine the distribution of dopamine (DA) axons after simultaneously confronting DA cells with both target cells of the corpus striatum (CS) and with non-target cells of the tectum (T). Dissociated fetal cells of rostral mesencephalic tegmentum (RMT) containing DA neurons and dissociated non-target tectal cells were exposed to wheat germ agglutinin conjugated to the fluorescent dye, rhodamine, prior to reaggregation in rotatory culture in order to distinguish these cells from non-dyed CS cells in the resulting reaggregates. After 9 days in culture, RMT-T-CS reaggregates were exposed to 10(-5)M DA and processed for DA-fluorescence histochemistry. Single reaggregates were serially sectioned and color photomicrographs prepared from each section. It was found that non-target cells (red rhodamine fluorescence) segregated from the undyed target cells, forming discrete areas containing the two cell types. DA nerve cell bodies and their processes could be distinguished by their green fluorescence. Since it is not possible to distinguish axonal from dendritic processes in a single section, the extent of neuronal dendritic arborization was estimated from reaggregates prepared from cells of the RMT and non-target T cells, in which there is no extensive proliferation of DA axons and fluorescent DA processes (presumably dendrites) are confined to the area near the cell body. Following exclusion of DA dendritic fluorescence, it was found that 85.2% of the presumed axonal fibers were present in the non-dyed areas containing striatal target cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Recent studies suggest that nerve growth factor is present within the central nervous system where it may exert selective trophic effects on cholinergic neurons. We have measured the effects of nerve growth factor on septal cholinergic neurons in three-dimensional reaggregating cell cultures, a system which closely simulates the cellular environment in situ. Septal cells obtained from 15-day-old mouse embryos were dissociated into a single cell suspension and then allowed to reaggregate in culture in a rotary incubator shaker. After 17 days in culture, half of the reaggregates from a flask were sonicated for measurement of choline acetyltransferase activity, and the remaining reaggregates were processed for acetylcholinesterase histochemistry. Addition of nerve growth factor to medium containing septal reaggregates resulted in greater than a three-fold increase in choline acetyltransferase activity and in the number of acetylcholinesterase-positive cells, as well as an enhancement in the staining of acetylcholinesterase-positive fibers. All of these effects of nerve growth factor could be neutralized by antibodies to nerve growth factor. In order to evaluate the possible role of endogenous hippocampal-derived nerve growth factor, antiserum to nerve growth factor was added to the culture media containing septal-hippocampal coaggregates. After 21 days in culture, the presence of nerve growth factor antibodies did not qualitatively affect the pattern or density of cholinergic fibers observed. Synapse formation between cholinergic axons and hippocampal target cells was still in evidence as revealed by electron microscopy. However, there was a modest decrease in choline acetyltransferase activity (20%) and cholinergic cell number (30%) when compared with coaggregates grown in culture medium either without nerve growth factor antiserum or with non-immune serum. The magnitude of these effects was markedly less than the effects observed when exogenous nerve growth factor was added to septal cells grown alone in reaggregate culture. These results suggest that nerve growth factor may play a role during central cholinergic development, but that additional trophic mechanisms are likely to be required.

An antibody to tyrosine hydroxylase has been used in a correlated light and electron microscopic study to characterize dopaminergic neurons and synaptic junctions in three-dimensional reaggregate cell culture. Dissociated fetal mesencephalic cells containing dopamine neurons were coaggregated with dissociated fetal striatal cells in rotatory culture for 21 days. Sections of the coaggregates were stained by the peroxidase anti-peroxidase technique to reveal tyrosine hydroxylase-immunoreactive structures. Clusters of immunoreactive perikarya as well as dendrites and axons were observed. Immunolabeled perikarya were round or oval and approximately 20 microns in diameter. Boutons immunoreactive for tyrosine hydroxylase formed symmetric synapses, primarily with unlabeled dendritic shafts. Symmetric membrane specializations were also observed between tyrosine hydroxylase-positive boutons and unlabeled dendritic spines as well as with the perikaryon of an unlabeled medium-size neuron possessing a slightly indented nucleus. To characterize the neurochemical nature of the neurons postsynaptic to tyrosine hydroxylase-positive boutons in the reaggregates, an antibody against DARPP-32 (a dopamine and adenosine 3':5'-monophosphate-regulated phosphoprotein) and an antibody against tyrosine hydroxylase were employed to visualize striatal dopaminoceptive neurons and dopaminergic structures, respectively, in the same section. Examination of reaggregate sections at the light microscopic level demonstrated that DARPP-32-immunoreactive cells were distributed into discrete clusters that were associated with patches of tyrosine hydroxylase-positive axonal varicosities. Ultrastructural analysis of tyrosine hydroxylase-positive boutons in such clusters revealed that dopaminergic axons synaptically contacted DARPP-32-immunoreactive neurons as well as unlabeled neuronal structures.

We have previously demonstrated at the light microscopic level that when embryonic day-15 septal neurons are co-cultured for 21 days with their target cells from the hippocampus, increased numbers of septal cholinergic neurons are present as compared with co-cultures employing cells from the non-target cerebellum. In addition, fine varicose axon-like cholinergic fibers are found to be associated with the hippocampal cells but not with cerebellar cells. We now provide ultrastructural evidence for hippocampal target cell-enhanced cholinergic neuronal survival, axonal proliferation, and synapse formation in this culture system. Dissociated cell suspensions from septal, hippocampal, and cerebellar areas were obtained from 15-day mouse embryos; and hippocampal and cerebellar cells were internally labeled with rhodamine-conjugated wheat germ agglutinin. Combinations of septal and hippocampal cells, and septal and cerebellar cells were allowed to reaggregate in rotation mediated culture for either 15 or 21 days. The reaggregates were then fixed, embedded, sectioned, and processed for acetylcholinesterase histochemistry. Sections were examined microscopically under bright-field optics to identify acetylcholinesterase-positive cells and fibers, and under fluorescence to locate rhodamine-labeled cell populations. Representative reaggregate profiles were then re-embedded for electron microscopic examination. In both types of reaggregates, either labeled hippocampal target or cerebellar non-target cells segregated from the septal cells so that areas containing each of the respective cell populations could be studied. In sections of septal-hippocampal reaggregates from 15-day cultures, 571 out of 665 (85%) cholinergic neurons examined were intact, whereas 15% of the cells showed some ultrastructural features of degeneration. Similarly, at day 21, 297 out of 335 (88%) of the cholinergic neurons were intact. In sections of septal-cerebellar reaggregates from 15-day cultures, 473 out of 572 (83%) cholinergic neurons were intact. By day 21 of culture, however, only 15 out of 110 (14%) cholinergic neurons examined were intact from the septal-cerebellar reaggregates. In areas of septal-hippocampal reaggregates occupied by rhodamine-labeled hippocampal cells, profiles of acetylcholinesterase-labeled axons were identified, and synaptic specializations were observed between cholinergic terminals and dendrites as well as somata of hippocampal target cells. In contrast, areas of septal-cerebellar reaggregates occupied by rhodamine-labeled cerebellar cells were devoid of cholinergic fibers. In areas of the septal-cerebellar reaggregate sections containing cholinergic fibers, many of such fibers exhibited degenerative changes.

Three-dimensional, rotation-mediated, reaggregate tissue cultures formed from dissociated fetal rostral mesencephalic tegmental (RMT) and corpus striatal (CS) or frontal cortical (FCx) cells were used to study methamphetamine neurotoxicity. Analysis of dopamine (DA), serotonin (5-HT) and gamma-aminobutyric acid (GABA) levels using HPLC techniques revealed decreases in RMT-CS and RMT-FCx reaggregate DA and 5-HT levels after treatment between 14 and 21 days in culture with methamphetamine in concentrations ranging from 10(-6)M to 10(-3)M. Dopamine cell numbers in RMT-CS and RMT-FCx reaggregates were estimated after visualization by histofluorescent techniques. Methamphetamine treatment caused decreases in DA cell numbers which paralleled the decreases in endogenous DA levels. Estimates of the accumulation of exogenous DA by RMT-CS reaggregates treated with methamphetamine showed that the amount of accumulation per cell remained fairly constant despite marked reductions in total DA cell numbers. This suggests that the reductions in endogenous DA levels following methamphetamine were secondary to loss of entire DA neurons rather than of a portion of the terminal axonal fields in the surviving neurons. Reaggregate tissue cultures are a useful tool in the study of potential neurotoxic effects of new or untested psychotherapeutic agents.

The influence of hippocampal target cells on the development of cholinergic septal neurons was studied in rotation-mediated reaggregating cell cultures. Brain cells from 15-day-old mouse embryos were obtained from:(i)septum, containing cholinergic cells which project to the hippocampus;(ii)hippocampus which contains target cells for the septal cholinergic neurons; and(iii)cerebellum, containing cells which are not targets for the septal cholinergic cells. The cells were then cultured for 3 weeks in a rotary incubator in the following combinations:(a)septal cells alone;(b)hippocampal cells alone;(c)cerebellar cells alone;(d)septal-hippocampal cells together; and(e)septal-cerebellar cells together. After harvesting, fixation, and embedding, 50 μm sections were cut and processed for visualization of acetylcholinesterase activity. Sections from reaggregates containing either hippocampal or cerebellar cells alone contained only a few acetylcholinesterase-positive cells, but no positive fibers. Sections from septal-hippocampal coaggregates revealed a pattern of well-defined, fine-caliber acetylcholinesterase-positive fibers with extensive arborizations and varicosities suggesting axonal proliferation. In septal-cerebellar coaggregates, acetylcholinesterase-positive fibers appeared to be degenerating and distinct areas were observed which were essentially devoid of acetylcholinesterase fibers. In some experiments, either cerebellar or hippocampal cells were labeled with wheatgerm agglutinin-rhodamine prior to culture in order to identify these cells in the resulting reaggregates. Analysis of sections from these studies showed that acetylcholinesterase fibers were excluded from regions of coaggregates containing cerebellar cells, but were present in regions of coaggregates containing hippocampal cells. Finally, cell counts of acetylcholinesterase-positive cells in the various combinations revealed that these putative cholinergic neurons were significantly more numerous in septal-hippocampal coaggregates (271 ± 19 per 106 septal cells added) than in septal reaggregates (38 ± 6 per 106 septal cells added) or septal-cerebellar coaggregates (85 ± 29 per 106 septal cells added). These results, taken together, suggest that hippocampal target cells influence the development and survival of cholinergic neurons.

Spontaneous release of [3H]dopamine (DA) was observed from reaggregates of dissociated cells from fetal rostral mesencephalic tegmentum (RMT) containing DA neurons cocultured with their axonal target cells from striatum (CS) or frontal cortex (FCx), or with non-target cells from occipital cortex (OCx), or tectum. Such release increased in response to 50 mM K+. Tetrodotoxin (TTX) suppressed the spontaneous release from RMT-CS and RMT-FCx reaggregates by 42%; from RMT-tectum reaggregates by 24%, and did not significantly inhibit the release from RMT-OCx cocultures. Since TTX blocks spontaneous neuronal activity, these results suggest that the presence of axonal target cells enhances the activity of the dopamine neurons. DA neurons within RMT-FCx reaggregates released significantly more [3H]DA in response to 50 mM K+ than in RMT-CS cocultures. This result is in accord with the findings in vivo that inhibitory feedback mechanisms on DA release, present in the striatum, are lacking in the frontal cortex.

Dissociated, 14-day-old embryonic cells of the rostral mesencephalic tegmentum (RMT), including the dopamine neurons of this region, were allowed to reaggregate and develop in rotatory culture for 7 days in the presence of dissociated embryonic cells from the target areas of the dopaminergic neurons, corpus striatum (CS) or frontal cortex (FCx). Alternatively, RMT cells were allowed to reaggregate by themselves or in the presence of dissociated cells from a telencephalic area, occipital cortex (OCx), or mesencephalic area, tectum (T), which are not target areas for the dopamine neurons. Histofluorescence analysis revealed the number of dopamine neurons contained within reaggregates of any given type. Approximately 4 times as many dopamine neurons were found in RMT-CS coaggregates and 1.5 times as many in RMT-FCx coaggregates than in aggregates constituted from cells of the RMT either alone, or in coaggregates from RMT-OCx or RMT-T. Since axonal process formation and maintenance can only be observed in RMT-CS and RMT-FCx coaggregates, the enhanced dopamine neuron survival is probably due to an interaction of dopaminergic axonal processes with target cells within the reaggregates.

Dissociated dopamine (DA) neurons from 14-day fetal mice were dissected from the rostral mesencephalic tegmentum (RMT) and were allowed to reaggregate in vitro with cells from the corpus striatum (CS). As previously demonstrated under these conditions, DA neurons develop punctate fluorescent varicosities and the capacity to synthesize, accumulate, and retain DA (Kotake, C., P. C. Hoffmann, and A. Heller (1982) J. Neurosci. 2: 1307–1315). After 17 to 22 days in culture, the RMT-CS coaggregates were assessed for their ability to release DA. Coaggregates were incubated in 5.6 x 10(-6) M [3H]DA, washed, and then superfused at 100 microliters/min for 2 hr. Fractions were collected every 2 min. Basal efflux of [3H]DA/2 min was 1% of tissue stores of 3H. K+, 70 mM infused for 8 min induced a peak release of 5.87% of tissue stores of 3H, and 50 mM K+ induced a peak release of 2.13%. The potassium-induced release of [3H]DA was calcium dependent. When d- amphetamine was infused for 12 min, 100 microM solutions induced a peak release of 8.91%, 10 microM induced a peak release of 4.36%, and 1 microM induced a peak release of 1.85% of tissue stores of 3H. Substance P at 100 microM induced a peak release of 2.19% of tissue stores of 3H. Tetrodotoxin (0.5 and 2.5 microM) decreased basal efflux by 40%, blocked substance P-induced release, but did not affect either potassium- or d-amphetamine-induced release of [3H]dopamine.

Previous studies from this laboratory on the functional ontogeny of impulse flow in the nigrostriatal pathway of the neonatal rat were interpreted as indicating that there was an abrupt onset of impulse flow between 6 and 8 days of age. The functional development was assessed by determining the age at which the biochemical responses of these neurons to physical or pharmacological manipulation were similar to those of adult neurons. Further studies of these phenomena demonstrate that the age-dependency of these biochemical markers of impulse traffic are directly related to the development of thermoregulatory function in the neonatal rat. Accordingly, provided that 4-day-old or even younger rats are maintained at or near litter temperature, they exhibit qualitatively all of the biochemical responses characteristic of the 10-day-old or adult animal in which impulse flow is known to be present.