Valeria Franceschini

Cochlear Repair by Transplantation of Human Cord Blood CD133+ Cells to Nod-Scid Mice Made Deaf with Kanamycin and Noise

We investigated the fate of human cord blood CD133+ hematopoietic stem cells (HSC) transplanted intravenously (IV) into irradiated nodscid mice previously made deaf by ototoxic treatment with kanamycin and/or intense noise, to verify whether HSC engraft the cochlea and contribute to inner ear restoration, in vivo. We tested the presence of HLA.DQα1 by PCR, used for traceability of engrafted cells, finding evidence that HSC migrated to various host tissues, including the organ of Corti (OC). By histology, antibody and lectin-staining analysis, we confirmed that HSC IV transplantation in mice previously damaged by ototoxic agents correlated with the repair process and stimulation ex novo of morphological recovery in the inner ear, while the cochlea of control oto-injured, nontransplanted mice remained seriously damaged. Dual color FISH analysis also provided evidence of positive engraftment in the inner ear and in various mouse tissues, also revealing small numbers of heterokaryons, probably derived from fusion of donor with endogenous cells, for up to 2 months following transplantation. These observations offer the first evidence that transplanted human HSC migrating to the inner ear of oto-injured mice may provide conditions for the resumption of deafened cochlea, emerging as a potential strategy for inner ear rehabilitation.

Glial fibrillary acidic protein and vimentin immunoreactivity of astroglial cells in the central nervous system of adult Podarcis sicula (Squamata, Lacertidae)

The present immunoperoxidase cytochemical study describes the distribution of glial intermediate filament molecular markers, glial fibrillary acidic protein (GFAP) and vimentin, in the brain and spinal cord of the adult lizard, Podarcis sicula. GFAP immunoreactivity is abundant and the positive structures are mainly represented by fibres of different lengths which are arranged in a rather regular radial pattern throughout the CNS. They emerge from generally immunopositive radial ependymoglia and are directed from the ventricular wall towards the meningeal surface. The glial fibres give origin to endfeet which are apposed to the blood vessel walls and subpial surface where they form the continous perivascular and subpial glia envelopes, respectively. In the optic tectum and spinal cord, star‐shaped astrocytes coexist with radial glia. In the spinal cord, cell bodies of immunopositive radial glia are displaced from the ependyma. While vimentin immunoreactive elements are almost completely absent in the brain except for a few diencephalic radial fibres, the spinal cord ependyma exhibits a clearly vimentin positivity and no GFAP staining. In the Podarcis CNS the immunocytochemical response of the astroglial intermediate filaments appears typical of mature astroglia cell lineage since it fundamentally expresses GFAP immunoreactivity. Moreover, this immunocytochemical study shows that the Podarcis fibre pattern with predominant radial glial cells is morphologically more immature than in avians and mammalians, a condition suggesting that reptiles represent a fundamental step in the phylogenetic evolution of vertebrate astroglial cells.

Identification of surface glycoconjugates in the olfactory system of turtle

Lectin binding histochemistry was performed on the olfactory system of Pseudemys scripta to investigate the distribution and density of defined carbohydrate terminals on the cell surface glycoproteins of the olfactory receptors and their terminals in the olfactory bulbs. The lectin staining patterns indicate that the receptor cells of the olfactory mucosa are characterized by glycoconjugates containing alpha-D-galactose and N-acetyl-D-glucosamine terminal residues. The vomeronasal receptor cells contain instead alpha-N-acetyl-D-galactosamine, N-acetyl-D-glucosamine and alpha-D-galactose residues. The results demonstrate that the vomeronasal receptor cells contain high density of alpha-N-acetyl-D-galactosamine sugar residues that are not expressed by receptor cells of the olfactory mucosa. The presence of specific glycoproteins, whose terminal sugars are detected by lectin binding, might be related to the chemoreception and transduction of the odorous message into a nervous signal or in the histogenesis of the olfactory system. In fact, the olfactory receptors are the only known neurons in the vertebrate nervous system that undergo a continual cycle of proliferation not only in developing animals but also in mature ones. Moreover the results show that BSA-I-B4, an alpha-D-galactosyl-specific isolectin, targets the terminal sugar residues in the ramified microglial cells.

Histochemical study by lectin binding of surface glycoconjugates in the developing olfactory system of rat

Lectin‐binding histochemistry was used to investigate the distribution and density of defined carbohydrate sequences on the cell surface glycoproteins of the olfactory receptors of rat during development. The olfactory and vomeronasal receptors showed a positive labelling after biotinylated Lycopersicum esculentum lectin binding on embryonic day 16 (E16), while horseradish peroxidase‐labelled Glycine max, Bandeiraea simplicifolia (BS A‐I) and its B4 isomer BSA‐I‐B4 agglutinins started to label from day 18 (E18). From this stage onward there was a progressive increase in the intensity and number of lectin‐binding olfactory receptors. The first lectin‐labelled bundles of axons penetrating the olfactory bulb were observed on E20; from E21 it was possible to identify the first labelled glomeruli that, on the first day (P1) of postnatal life, showed a feature very similar to that of the adult. The lectin staining patterns indicate that during development there are differences in the kind and distribution of saccharidic moieties on the surface of rat olfactory neurons. The possible role of carbohydrate‐containing glycoproteins in the reception and transduction of the odours and in the modulation of the cell‐cell interactions in the olfactory system is discussed.

Lectin cytochemical localisation of glycoconjugates in the olfactory system of the lizards Lacerta viridis and Podarcis sicula

To investigate the presence of defined carbohydrate moieties on the cell surface of the olfactory and vomeronasal receptor cells and the projections of the latter into the olfactory bulbs, a lectin binding study was performed on the olfactory system of the lizards: Lacerta viridis and Podarcis sicula. Both lizards showed a high lectin binding for N-acetyl-glucosamine in the sensory neurons. The lectin binding patterns in Lacerta indicated that the main olfactory system possessed a moderate density of N-acetyl-galactosamine residues and detectable levels of galactose ones. The vomeronasal system on the other hand contained a high density of N-acetyl-galactosamine moieties and a moderate density of glucosamine ones. In Podarcis the main olfactory system and vomeronasal organ contained respectively detectable and moderate levels of galactose residues. The expression of specific glycoconjugates may be associated with outgrowth, guidance and fasciculation of olfactory and vomeronasal axons.

Immunohistochemical and Histochemical Characteristics of the Olfactory System of the Guppy, Poecilia reticulata (Teleostei, Poecilidae)

Olfaction in fish has been studied using preferentially macrosmatic species as models. In the present research, the labelling patterns of different neuronal markers and lectins were analyzed in the olfactory neurons and in their bulbar axonal endings in the guppy Poecilia reticulata, belonging to the group of microsmatic fish. We observed that calretinin immunostaining was confined to a population of olfactory receptor cells localized in the upper layers of the sensory mucosa, probably microvillous neurons innervating the lateral glomerular layer. Immunoreactivity for S100 proteins was mainly evident in crypt cells, but also in other olfactory cells belonging to subtypes projecting in distinct regions of the bulbs. Protein gene product 9.5 (PGP 9.5) was not detected in the olfactory system of the guppy. Lectin binding revealed the presence of N‐acetylglucosamine and α‐N‐acetylgalactosamine residues in the glycoconjugates of numerous olfactory neurons ubiquitously distributed in the mucosa. The low number of sugar types detected suggested a reduced glycosidic variability that could be an index of restricted odorant discrimination, in concordance with guppy visual‐based behaviors. Finally, we counted few crypt cells which were immunoreactive for S100 and calretinin. Crypt cells were more abundant in guppy females. This difference is in accordance with guppy gender‐specific responses to pheromones. Cells immunoreactive to calretinin showed no evidence of ventral projections in the bulbs. We assumed the hypothesis that their odorant sensitivity is not strictly limited to pheromones or sexual signals in general. Anat Rec, 2009.

Glial cytoarchitecture in the central nervous system of the soft-shell turtle, Trionyx sinensis, revealed by intermediate filament immunohistochemistry

The distribution of the intermediate filament molecular markers, glial fibrillary acidic protein (GFAP) and vimentin, has been studied in the central nervous system (CNS) of the soft-shell turtle (Trionyx sinensis) with immunoperoxidase histochemistry. GFAP immunohistochemistry pointed out the presence of different astroglial cell types. The brain pattern consists of ependymal radial glia whose cell bodies are located in the ependymal layer throughout the brain ventricular system. In the spinal cord, the ependyma is immunonegative, whereas positive radial astrocyte cell bodies are displaced from the ependyma into the periependymal position. Star-shaped astrocytes are observed only in the posterior intumescence of the spinal cord. The different regions of the CNS show a different intensity in GFAP immunostaining even in the same cellular type. Vimentin-immunoreactive structures are absent in the brain and spinal cord. The present study reports an heterogeneous feature of the astroglial pattern in the spinal cord compared to the brain which shows an ancestral condition.

Lectin-Binding Patterns in the Olfactory System of the Lizard, Physignathus lesueurii

Lectin binding histochemistry was performed on the olfactory system of Physignathus lesueurii to investigate the distribution and density of defined carbohydrate terminals on the cell‐surface glycoproteins of the olfactory and vomeronasal receptor cells and their terminals in the olfactory bulbs. The lectin staining patterns indicate that the vomeronasal and olfactory receptor cells are characterized by glycoconjugates containing α‐D‐galactose and N‐acetyl‐D‐glucosamine terminal residues. The presence of specific glycoproteins, whose terminal sugars are detected by lectin binding, might be related to the chemoreception and transduction of the odorous message into a nervous signal or to the histogenesis and development of the olfactory system. The olfactory and vomeronasal receptor cells are vertebrate neurons that undergo a continual cycle of proliferation not only during development but also in mature animals. J. Morphol. 247:34–38, 2001.

Human Cord Blood CD133+ Stem Cells Transplanted to Nod-Scid Mice Provide Conditions for Regeneration of Olfactory Neuroepithelium After Permanent Damage Induced by Dichlobenil†‡

The herbicide dichlobenil selectively causes necrosis of the dorsomedial part of olfactory neuroepithelium (NE) with permanent damage to the underlying mucosa, whereas the lateral part of the olfactory region and the nasal respiratory mucosa remain undamaged. We investigated here whether human umbilical cord blood CD133+ stem cells (HSC) injected intravenously to nod‐scid mice pretreated with dichlobenil may engraft the olfactory mucosa and contribute to the regeneration of the damaged NE. We tested HLA‐DQα1 DNA and three human microsatellites (Combined DNA Index System) as indicators of engrafted cells, finding polymerase chain reaction evidence of chimaerism in various tissues of the host, including the olfactory mucosa and bulb, at 7 and 31 days following HSC transplantation. Histology, immunohistochemistry, and lectin staining revealed the morphological recovery of the dorsomedial region of the NE in dichlobenil‐treated mice that received HSC, contrasting with the lack of regeneration in similarly injured areas as these remained damaged in control nontransplanted mice. FISH analysis, to detect human genomic sequences from different chromosomes, confirmed persistent engraftment of the regenerating olfactory area with chimeric cells. Electro‐olfactograms in response to odorants, to test the functionality of the olfactory NE, confirmed the functional damage of the dorsomedial area in dichlobenil‐treated mice and the functional recovery of the same area in transplanted mice. These findings support the concept that transplanted HSC migrating to the damaged olfactory area provide conditions facilitating the recovery from olfactory receptor cell loss. STEM CELLS 2009;27:825–835

Age-related impairment of olfactory bulb neurogenesis in the Ts65Dn mouse model of Down syndrome.

Down syndrome (DS) is a genetic condition caused by triplication of chromosome 21. Widespread neurogenesis reduction during brain development underlies the numerous neurological defects of DS. These defects start to manifest themselves at birth and worsen with age. However, unlike other brain functions, smell is impaired only at advanced life stages, suggesting preservation of olfactory bulb neurogenesis up to adulthood. To clarify this issue, in the current study we examined olfactory bulb (OB) neurogenesis and olfactory function by exploiting the Ts65Dn mouse, a widely used model of DS. We found that in young (15-day-old) Ts65Dn mice, in spite of a reduced proliferation rate in the subventricular zone (SVZ) in comparison with euploid mice, the number of neuroblasts traveling in the rostral migratory stream (RMS), en route to the OB, and the number of new granule neurons added to the OB were similar to those of euploid mice. In mid-age (13-month-old) Ts65Dn mice, however, the proliferation rate in the SVZ was more severely reduced in comparison with euploid mice and the number of neuroblasts in the RMS and new granule neurons added to the OB underwent a reduction. While in young Ts65Dn mice the olfactory function, assessed with the buried food pellet test, was similar to that of euploid mice, in mid-age mice it was significantly impaired. Taken together, results suggest that an age-related reduction in the renewal of OB granule cells may underlie the age-related smell impairment in DS.