Prevalence and evaluation of oropharyngeal dysphagia in patients with severe acute respiratory syndrome coronavirus 2 infection in the intensive care unit.

OBJECTIVE: The main objective was to assess the prevalence of dysphagia in the intensive care unit in patients with coronavirus disease 2019.Methods. A cohort, observational, retrospective study was conducted of patients admitted to the intensive care unit for severe acute respiratory syndrome coronavirus 2 pneumonia at the University Hospital of Rouen in France. RESULTS: Over 4 months, 58 patients were intubated and ventilated, 43 of whom were evaluated. Screening revealed post-extubation dysphagia in 62.7 per cent of patients. In univariate analysis, a significant association was found between the presence of dysphagia and: the severity of the initial pathology, the duration of intubation, the duration of curare use, the degree of muscle weakness and the severity indicated on the initial scan. At the end of intensive care unit treatment, 22 per cent of the dysphagic patients had a normal diet, 56 per cent had an adapted diet and 22 per cent still received exclusive tube feeding. CONCLUSION: Post-extubation dysphagia is frequent and needs to be investigated.

Pax2, Otx2, Gbx2 and Fgf8 expression in early otic vesicle development.

The inner ear is a suitable system to study the mechanisms involved in the specification of different functional domains during morphogenesis. Using single and double in situ hybridization (ISH) we show that three transcription factors (Otx2, Gbx2and Pax2) and a member of the fibroblast growth factor family (Fgf8) could participate in the compartmentalization of the otic vesicle and in the formation of the acoustic-vestibular ganglion.

[Formation of the boundary between the midbrain and the hindbrain: involvement of Otx2 and Gbx2 genes]. / Formation de la frontière entre cerveaux moyen et postérieur: implication des gènes Otx2 et Gbx2.

We have studied the neuromeric organisation of the mesencephalic-metencephalic (mes-met) territory of the avian neural tube using chick/quail transplantation experiments and analysing the expression of various regulatory genes in chimeric and normal embryos. Homotopic grafts demonstrate the presence of an interneuromeric boundary separating the mesencephalic and cerebellar territories (the mes-met or midbrain/hindbrain boundary). This boundary is characterised from HH10 onwards by the confrontation of the Otx2-Wnt1 and Gbx2-Fgf8 expressing domains, while En2 and Pax2 genes are expressed at both sides of the mes-met boundary. The evolution of the position of the Otx2/Gbx2 boundary with respect to the vesicles and constriction observed within the mes-met domain between stages HH10 and HH20, allows us to redefine the fate map of this region and to propose a new nomenclature for HH10. Transplantation between the prosencephalic neuroepithelium and the mes-met domain shows the possibility of inducing a mes-met phenotype within the two caudal-most prosomeres, preceded by its characteristic genetic cascade. The induction selectively takes place along the boundary between the graft (Otx2 positive) and the host cerebellar territory (expressing high levels of Gbx2); this includes the induction inside the graft of a new Otx2/Gbx2 boundary. Conversely, no induction is ever observed when the graft is confronted to the host Otx2 expressing domain. Although Fgf8 may be involved in the inductive events, our data strongly suggest that confrontation between Otx2 and Gbx2 is essential as an organiser of the mes-met domain.

Fgf8 and Gbx2 induction concomitant with Otx2 repression is correlated with midbrain-hindbrain fate of caudal prosencephalon.

Chick/quail transplantation experiments were performed to analyse possible factors involved in the regionalisation of the midbrain-hindbrain domain. The caudal prosomeres, expressing Otx2, were transplanted at stage HH10 into rostrocaudal levels of the midbrain-hindbrain domain, either straddling the intra-metencephalic constriction (type 1 grafts), or at rostral and medial levels of pro-rhombomere A1 (type 2 and 3 grafts, respectively); thus, in all situations, one border of the graft was in contact with the host Gbx2- and Fgf8-expressing domains. The area containing the graft, recognised by QCPN immunohistochemistry, was first analysed 48 hours after transplantation for Otx2, Gbx2, En2 and Fgf8. Although in all three situations, a large part of the graft maintained Otx2 expression, another part became Otx2 negative and was induced to express Gbx2 and Fgf8. These inductive events occurred exclusively at the interface between the Otx2-positive transplanted domain and the ipsilateral host Gbx2-positive rhombomere 1, creating a new Otx2-Gbx2 boundary within the grafted territory. In type 1 and 2 grafts, the induced Fgf8 domain is in continuity with the host Fgf8 isthmic domain, whereas for type 3 grafts, these two domains are separate. High levels of En2 expression were also induced in the area expressing Gbx2 and Fgf8, and Wnt1 and Pax2 expressions, analysed in type 3 grafts, were induced at the intragraft Otx2-Gbx2 new boundary. Moreover, at later embryonic stages, the graft developed meso-isthmo-cerebellar structures. Thus, gene expressions induced in the grafted prosencephalon not only mimicked the pattern observed in the normal midbrain-hindbrain domain, but is followed by midbrain-hindbrain cytodifferentiation, indicating that not only Fgf8 but also confrontation of Otx2 and Gbx2 may play an essential role during midbrian-hindbrain regionalisation.

Comparative analysis of Otx2, Gbx2, Pax2, Fgf8 and Wnt1 gene expressions during the formation of the chick midbrain/hindbrain domain.

The patterns of the Gbx2, Pax2, Wnt1, and Fgf8 gene expression were analyzed in the chick with respect to the caudal limit of the Otx2 anterior domain, taken as a landmark of the midbrain/hindbrain (MH) boundary. The Gbx2 anterior boundary is always concomitant with the Otx2 posterior boundary. The ring of Wnt1 expression is included within the Otx2 domain and Fgf8 transcripts included within the Gbx2 neuroepithelium. Pax2 expression is centred on the MH boundary with a double decreasing gradient. We propose a new nomenclature to differentiate the vesicles and constrictions observed in the avian MH domain at stage HH10 and HH20, based on the localization of the Gbx2/Otx2 common boundary.

Cajal-Retzius cells regulate the radial glia phenotype in the adult and developing cerebellum and alter granule cell migration.

Studies on the reeler mutation have shown that pioneer Cajal-Retzius (CR) cells are involved in neuronal migration in the developing cortex. Here, we use grafting and coculture experiments to investigate the mechanisms by which CR cells govern migration. We show that transplantation of embryonic CR cells, but not other cortical neurons, into adult cerebella induces a transient rejuvenation of host Bergmann glia into a radial glia phenotype. Similarly, CR cells sustain the phenotype of developing radial glia in postnatal cerebellar slices and induce the organization of a glial scaffold inside the CR cell explants. Studies with semipermeable inserts show that these effects are mediated by diffusible signals. We also show that CR cells adjacent to the surface of cerebellar slices reverse the direction of the migration of granule cells. Finally, CR cells from reeler mutant embryos elicited similar effects. These observations imply a role for CR cells in the regulation of the radial glia phenotype, a key step for neuronal migration, and suggest that these pioneer neurons may also exert a chemoattractive influence on migrating neurons.

The chick/quail chimeric system: a model for early cerebellar development.

The chick/quail chimeric system is now extensively used to study the development of the central nervous system. Here we discuss data obtained by this powerful experimental approach by which to study several issues of the cerebellar ontogenesis. We first discuss experiments which have allowed redefinition of the localization of the cerebellar primordium in the early neural tube and which suggest that the cerebellum could originate from different morphogenetic units. Then, we discuss experiments testing the possible role of the homeobox containing gene En-2 in cerebellar specification and showing that the En-2 expressing cerebellar neuroepithelium can act as an organizer. Finally we discuss data obtained in chimeric embryos with partial cerebellar grafts used to reexamine the origin and settling of several types of cortical cerebellar cells, in particular granule cells, molecular layer interneurons and Purkinje cells.

The caudal limit of Otx2 gene expression as a marker of the midbrain/hindbrain boundary: a study using in situ hybridisation and chick/quail homotopic grafts.

Segmentation of the neural tube has been clearly shown in the forebrain and caudal hindbrain but has never been demonstrated within the midbrain/hindbrain domain. Since the homeobox-containing gene Otx2 has a caudal limit of expression in this region, we examined, mainly in chick embryos, the possibility that this limit could represent an interneuromeric boundary separating either two cerebellar domains or the mesencephalic and cerebellar primordia. In situ hybridisation with chick or mouse Otx2 probes showed the existence of a transient Otx2-negative area in the caudal mesencephalic vesicle, between stages HH10 and HH17/18 in chick, and at embryonic day 9.5 in mice. The first post-mitotic neurons of the mesencephalon sensu stricto, as labelled with an anti-beta-tubulin antibody, overlay the Otx2-positive neuroepithelium with a perfect match of the caudal limits of these two markers at all embryonic stages analysed (until stage HH20). Chick/quail homotopic grafts of various portions of the midbrain/hindbrain domain have shown that the progeny of the cells located in the caudal mesencephalic vesicle at stage HH10 are found within the rhombomere 1 as early as stage HH14. Furthermore, our results indicate that the cells forming the HH20 constriction (coinciding with the caudal Otx2 limit) are the progeny of those located at the caudal Otx2 limit at stage HH10 (within the mesencephalic vesicle). As a result, the Otx2-positive portion of the HH10 mesencephalic vesicle gives rise to the HH20 mesencephalon, while the Otx2-negative portion gives rise to the HH20 rostral rhombomere 1. Long-survival analysis allowing the recognition of the various grisea of the chimeric brains strongly supports the view that, as early as stage HH10, the caudal limit of Otx2 expression separates mesencephalic from isthmo/cerebellar territories. Finally, this study revealed unexpected rostrocaudal morphogenetic movements taking place between stages HH10 and HH16 in the mediodorsal part of the caudal Otx2-positive domain.

Further observations on the susceptibility of diencephalic prosomeres to En-2 induction and on the resulting histogenetic capabilities.

It has been previously shown by chick/quail heterotopic grafts that En-2 expression and a mesencephalic phenotype can be induced within the avian primordial prosencephalic vesicle, although the induction appeared restricted to the caudal forebrain. The present experiments were aimed at further analyzing the competence of the prosencephalic neuroepithelium. Different types of grafts were performed between chick and quail embryos: (i) caudal forebrain grafts positioned in the midbrain/hindbrain junction (the En-2-positive domain); (ii) En-2-positive grafts integrated at different levels of the forebrain. In both cases, the grafts were transplanted either with a normal orientation or after inversion of their rostro-caudal axis. The chimeric embryos were analyzed at stages HH19-24 for expression of En-2 and Pax-6 homeobox-containing genes, normally expressed in the meso-isthmo-cerebellar and prosencephalic domains, respectively. A cytoarchitectonic analysis of grafted and surrounding host tissue was also performed at later developmental stages in chimeric embryos with caudal forebrain grafts. Our results show that the caudal diencephalon, including the prospective territories for prosomeres 1 and 2, is competent to express En-2 when in close contact to the En-2 polarizing region, whereas the more rostral neuroepithelium, including the prospective territories for the third prosomere and telencephalon, does not change its fate under similar conditions. The ectopic-induced neuroepithelium can develop mesencephalon, but also isthmus and cerebellum according to its site of integration rostrally or caudally to the mesencephalic/isthmo-cerebellar boundary. Our data also show that within the competent diencephalon, the induced En-2 expression can be arrested at the P1/P2 interneuromeric boundary. This arrest appears to be directionally oriented as it only takes place when the induction is produced within prosomere 1 but not when it comes from prosomere 2. These data can be considered as resulting from either a possible oriented permissiveness of cells which form the boundary separating prosomeres 1 and 2, or of a different permissiveness of the cells composing these two caudal prosomeres.

Expression of the homeobox-containing gene En-2 during the development of the chick central nervous system.

The expression of the homeobox-containing gene En-2 was analysed with the monoclonal antibody 4D9 in the chick central nervous system throughout embryogenesis. Confirming previous studies, early expression of the En-2 protein [beginning at stage 9 of Hamburger and Hamilton (HH9)] is restricted to a portion of the neural tube containing the primordia of the cerebellum, the isthmic region and the mesencephalic grisea, and forms a double gradient decreasing both caudally and rostrally from a high point located around the midbrain-hindbrain constriction. This mes-isthmo-cerebellar region contains all the En-2-positive germinative cells and the great majority of the En-2-positive postmitotic neurons throughout embryogenesis. Nevertheless, as the postmitotic neurons appear, En-2 expression also occurs outside this region: in two columns of non-motoneuron cells in rhombomeres two to four (between HH20 and HH30) and, from HH24 onwards, throughout the grey matter of the lumbar and thoracic spinal cord, with the exception of the ventral motoneuron columns. Here, a detailed description of En-2 expression is provided for the mes-isthmo-cerebellar region at stages HH30-32 [embryonic day (E) 7], HH37 (E11) and HH46 (E21, hatching). This allows the visualization of cellular groups with heterogeneous patterns of En-2 expression, which are specific for each group in the intensity of En-2 expression, the distribution of the labelled cells and the temporal regulation of the gene. The use of tyrosine hydroxylase antiserum shows coexpression of the tyrosine hydroxylase enzyme and En-2 protein in the caudal part of the nuclei tegmenti pedunculo-pontinus, the area ventralis of Tsai and the substantia grisea centralis, but not in the locus coeruleus. In the cerebellum, the first expression, which is located in the deep nuclei and parasagittal bands of Purkinje cells, is down-regulated when the molecular layer interneurons and the granular cells begin to express the gene, at the end of embryogenesis. Finally, at hatching, En-2 expression permits the visualization in the cerebellum of a population of small En-2-negative cells located around the Purkinje cells that may correspond to those described in chick/quail chimaeras as having an origin different from that of the bulk of granular neurons.

Molecular plasticity of adult Bergmann fibers is associated with radial migration of grafted Purkinje cells.

Embryonic Purkinje cells (PCs) from cerebellar primordia grafted in adult pcd mutant cerebellum replace missing PCs of the host, and become synaptically integrated into the defective cerebellar circuit. This process of neuronal replacement starts with the invasion of grafted PCs into the host cerebellum, and their radial migration through its molecular layer. The present study is aimed at determining whether the glial axes for this migration are embryonic radial glial cells that comigrate with the grafted PCs, or adult Bergmann fibers of the host, transiently reexpressing the molecular cues needed for their guidance of the migration. Transplants from a transgenic mouse line (Krox-20/lacZ14) in which Bergmann fibers could be identified by lacZ expression reveal that, despite the presence of X-gal-stained Bergmann fibers in the graft remnants and of grafted PCs in the host molecular layer, all Bergmann fibers in the host cerebellum lack of beta-galactosidase activity. Thus, these migratory axes belong to the host, not to the donor. Transplants from normal isogenic mouse embryos show that during the radial migration of grafted PCs (7 d after grafting) the involved host Bergmann fibers reexpress nestin (identified with monoclonal antibody Rat-401 immunostaining), normally expressed only by immature Bergmann fibers. Five days later, when grafted PCs have arrested their migration, host Bergmann fibers again become Rat-401 negative. These results indicate that embryonic PCs can trigger in adult cerebellum the molecular changes necessary for their own migration and ultimate synaptic integration in the host cortical circuitry.

Chick/quail chimeras with partial cerebellar grafts: an analysis of the origin and migration of cerebellar cells.

Chick/quail chimeras with partial cerebellar grafts have been performed to obtain further information about the origin and migratory movements of cerebellar cortical neurons. The grafts were performed by exchanging between these two species a precise, small portion of the E2 cerebellar primordium, as defined in Martinez and Alvarado-Mallart (Eur. J. Neurosci. 1:549-560, 1989). All grafts were done unilaterally. The chimeric cerebella, fixed at various developmental stages, were analyzed in serial Feulgen-stained preparations to map the distribution of donor and host cells in the ependymal layer (considered to be reminiscent of the primary germinative neuroepithelium) and in the various cortical layers. In some of the oldest cases, we also used antiquail immunostaining to recognize quail cells. In the ependymal layer, it has been possible to conclude that each hemicerebellar primordium undergoes a morphogenetic rotation that changes its rostrocaudal axis to a rostromedio-caudolateral direction. However, important individual variations were observed among the chimeric embryos with respect to the ependymal area expected to be formed by donor cells. These variations cannot be explained solely on the basis of microsurgical procedure; however, they suggest the existence of important reciprocal interaction between host and grafted neuroepithelia. Therefore, it was not possible to draw a precise fate map of the E2 cerebellar primordium. Nevertheless, the dispersion of grafted cells in the cerebellar cortex, when compared to the real extent of the ependymal grafted area in each particular case, provided important data: (1) The external granular layer (EGL), the secondary germinative epithelium, seems not to originate exclusively from the "germinative trigone," as is usually considered the case. It emerges from a larger but restricted portion of the primary cerebellar matrix extending about the caudal fourth or third of the ventricular epithelium, as defined after its morphogenetic rotation. (2) The Purkinje cells (PCs) develop from all areas of the cerebellar epithelium. Although the distribution of donor PCs parallels the grafted ventricular layer mediolaterally, donor PCs extend more in the rostrocaudal dimension. The PC layer is formed mainly by donor cells in the lobules underlain by the grafted ependymal layer. However, donor PCs are also observed in cortical lobules surmounting the host ventricular layer. In these lobules, the donor PCs form clusters of various widths interrupting the host PCs. Reciprocally, clusters of host PCs are also found in the lobules formed mainly by donor PCs. The alternate small clusters of donor or host PCs are surrounded by Bergmann fibers of the other species' origin.(ABSTRACT TRUNCATED AT 400 WORDS)

Regional specification during cerebellar development.

At early phases of development (stage HH12 of chick and quail embryos) the rostral part of the cerebellar anlage extends into the caudal third of the mesencephalic vesicle. This region is characterized by its high level of expression of the Wnt-1 and engrailed genes, which encode, respectively, for a secreted molecule and for a homeodomain protein. In order to analyze the potentialities of various parts of the cerebellar anlage, pieces of quail or mouse embryonic neural tubes have been transplanted heterotopically in the prosencephalon of HH12 chick embryos. These experiments indicated that signals operating in the plane of the neural tube are still operating in the donor avian and murine embryos. The met- mes- and prosencephalic territories of HH12 chick embryos are still competent to change their fates under the influence of these signals. It was also found that the Wnt-1 expression domain, the caudal mesencephalic vesicle, has striking organizing properties until late stages of development in avian and rodent embryos. The exact role of Wnt-1 in these inductive interactions is still unknown. Wnt-1 influence on cell fate could be mediated by the engrailed genes. Observations suggesting a later role of en-2 in the regionalization of the cerebellar cortex are also reported.

Relationship between Wnt-1 and En-2 expression domains during early development of normal and ectopic met-mesencephalon.

Grafting a met-mesencephalic portion of neural tube from a 9.5-day mouse embryo into the prosencephalon of a 2-day chick embryo results in the induction of chick En-2 (ChickEn) expression in cells in contact with the graft (Martinez et al., 1991). In this paper we investigate the possibility of Wnt-1 being one of the factors involved in En-2 induction. Since Wnt-1 and En-2 expression patterns have been described as diverging during development of the met-mesencephalic region, we first compared Wnt-1 and En-2 expression in this domain by in situ hybridization in mouse embryos after embryonic day 8.5. A ring of Wnt-1-expressing cells is detected encircling the neural tube in the met-mesencephalic region at least until day 12.5. This ring consistently overlapped with the En-2 expression domain, and corresponds to the position of this latter gene's maximal expression. We subsequently studied ChickEn ectopic induction in chick embryos grafted with various portions of met-mesencephalon. When the graft originated from the level of the Wnt-1-positive ring, ChickEn induction was observed in 71% of embryos, and in these cases correlated with Wnt-1 expression in the grafted tissue. In contrast, this percentage dropped significantly when the graft was taken from more rostral or caudal parts of the mesencephalic vesicle. Taken together, these results are compatible with a prolonged role of Wnt-1 in the specification and/or development of the met-mesencephalic region, and show that Wnt-1 could be directly or indirectly involved in the regulation of En-2 expression around the Wnt-1-positive ring during this time. We also provide data on the position of the Wnt-1-positive ring relative to anatomical boundaries in the neural tube, which suggest a more general role for the Wnt-1 protein as a positional signal involved in organizing the met-mesencephalic domain.

Tangential neuronal migration in the avian tectum: cell type identification and mapping of regional differences with quail/chick homotopic transplants.

This paper is a sequel to a previous report, using quail/chick chimeras with partial tectal transplants, in which a tangential invasion of host (chick) tectal territories by cells originating in the quail graft was demonstrated. The cells displaying this secondary tangential migration appeared restricted to two strata (stratum griseum centrale (SGC) and stratum griseum et fibrosum superficiale (SGFS)). Here we describe the morphology of the tangentially displaced neurons, as well as their overall distribution in the host tectal lobe, by means of an antibody that specifically recognizes quail cells, staining them in a Golgi-like manner. Neurons that migrated into the SGC are identified as multipolar projection neurons, typical of this stratum. The majority of cells that migrated into the SGFS correspond to horizontal neurons, as was also corroborated by observations in Golgi-impregnated material. These horizontal cells are concentrated in laminae b, d and f, where their processes form well delimited axonal plexuses. In confirmation of previous results, SGC neurons have a limited range of migration, whereas SGFS cells translocate across much longer distances. In reconstructions of appropriate cases, a remarkable polarity was noted. Significant invasion of chick tectum by quail cells mostly occurred in the rostral half of the host tectum. The long-range migration of superficial horizontal cells frequently reached, but did not cross, the rostral tectal boundary. Conversely, tangential migration in the caudal half of the host tectum was scarce and coincided with a typical arrangement of quail-derived radial columns interdigited with chick-derived columns. These findings are discussed in relation to existing data on immature neuronal populations, molecular marker distribution and polarity of the avian optic tectum.

New insight on the factors orienting the axonal outgrowth of grafted Purkinje cells in the pcd cerebellum.

Despite Purkinje cell replacement, leading to the repair of the cortical circuit of the pcd mouse cerebellum grafted with E12 cerebellar primordium, the reestablishment of the corticonuclear projection only occurs for some Purkinje cells and in a small percentage of grafted mice. In order to assess the importance of: (1) competition between host and grafted deep nuclei, and (2) the distance between the implants and the host deep nuclei, new grafted experiments have been performed. In the latter, solid grafts were taken from E13 or E14 donor embryos after removal of the region containing the postmitotic deep nuclear neurons, and randomly positioned at various cerebellar depths. With cortical implants, the absence of donor nuclear neurons is not sufficient to allow the axons of the grafted Purkinje cells that have invaded the host molecular layer to escape the confinement of this layer. The molecular/granular layer interface appears as an almost impassable obstacle, and the granule cell layer as a nonpermissive milieu. With grafts located between the host deep nuclei and the 4th ventricle (deep grafts), the grafted Purkinje cells project massively to the host nuclei, but they are unable to leave the implant and, therefore, they are not integrated in the deficient cortical circuit. Finally, when the grafts positioned in the central white matter (intermediate grafts) disrupt the integrity of the host granule cell layer, some of the grafted Purkinje cells invade the host molecular layer, while most of them remain within the implant. Some axons of the cortically integrated Purkinje cells, using the nearby graft as a bridge, seem able to innervate the host deep nuclei. The latter, in addition, receive a massive projection from the nonintegrated Purkinje cells. These results emphasize the ability of grafted Purkinje cells to specifically innervate their target host neurons, when either there is proximity, or when a permissive microenvironment for their axonal outgrowth is created by embryonic grafted cortical cerebellar neurons, filling the gap between the molecular layer and the deep nuclei of the host.

The reconstruction of cerebellar circuits.

Repair of adult 'point-to-point' systems by neural grafting is possible only when grafted neurons succeed in synaptically replacing the host's missing neurons, thus re-establishing the anatomical and functional integrity of the impaired circuits. Grafting experiments carried out on the cerebellum of the adult pcd (Purkinje-cell-degeneration) mutant mouse (an animal model of hereditary degenerative ataxia) reveal that embryonic Purkinje cells, by some unknown sorting mechanism, selectively invade the deprived cerebellar cortex. These neurons migrate to their proper domains and, inducing axonal sprouting of specific populations of host neurons, they become integrated synaptically within the pcd cerebellar cortex. However, the re-establishment of the corticonuclear projection is achieved only rarely, and this is the current experimental limit for the complete reconstruction of the cerebellar circuit.