Almudena Velasco

• University of Salamanca

The visual system of teleost fish grows continuously, which is a useful model for studying regeneration of the central nervous system. Glial cells are key for this process, but their contribution is still not well defined. We followed oligodendrocytes in the visual system of adult zebrafish during regeneration of the optic nerve at 6, 24, and 72 ho...

Zebrafish (Danio rerio) present continuous growth and regenerate many parts of their body after an injury. Fish oligodendrocytes, microglia and astrocytes support the formation of new connections producing effective regeneration of the central nervous system after a lesion. To understand the role of oligodendrocytes and the signals that mediate reg...

Objectives: To evaluate the impact of a quality-of-care improvement program implemented in emergency departments (EDs) in a Spanish autonomous community with the aim of reducing the use of unrecommended drugs when treating infants for acute bronchiolitis. Material and methods: Before-after quasi-experimental intervention study. We retrospectivel...

Oligodendrocytes are the myelinating cells in the central nervous system. In birds and mammals, the oligodendrocyte progenitor cells (OPCs) originate in the preoptic area (POA) of the hypothalamus. However, it remains unclear in other vertebrates such as fish. Thus, we have studied the early progression of OPCs during zebrafish visual morphogenesis...

Introduction: Resistance to antibiotics is a growing problem with repercussions on the choice of first-line treatment in urinary tract infection (UTI) in childhood. Objectives: To know the current pattern of antibiotic susceptibility/resistance of the most frequent germs that cause UTI in our healthcare area. Secondary objective is to know the e...

Apicobasal polarity is a hallmark of retinal pigment epithelium cells and is required to perform their functions; however, the precise roles of the different proteins that execute polarity are still poorly understood. Here, we have studied the expression and location of Scribble, the core member of the polarity basal protein complex in epithelial-d...

Abstract: Oligodendrocytes are the myelinating cells in the central nervous system (CNS). Their developmental origin and specification are well known in birds and mammals but remains unclear in fish. To fulfill this gap, we have studied their early progression during zebrafish visual morphogenesis using the transgenic line Olig2:GFP. We have track...

Simple Summary Doublecortin (DCX) is an essential protein in the development of the central nervous system and in lamination of the mammalian cortex. It is known that the expression of DCX is restricted to newborn neurons. The visual system of teleost fish has been postulated as an ideal model since it continuously grows throughout the animal’s lif...

Organization of a Sox2-positive glial cell population in the optic nerve associated with growing fibers in the fish visual system.

Apicobasal polarity is essential for epithelial cell function, yet the roles of different proteins in its completion is not fully understood. Here, we have studied the role of the polarity protein, CRB2, in human retinal pigment epithelial (RPE) cells during polarization in vitro, and in mature murine RPE cells in vivo. After establishing a simplif...

Purpose: To evaluate the therapeutic effect of subconjunctival injection of human mesenchymal stromal cells (hMSCs) in the cornea of mice with graft versus host disease (GVHD). Methods: GVHD was induced in mice after hematopoietic stem cell transplantation (HSCT) between MHC-mismatched mouse strains. Subconjunctival injection of hMSCs was applie...

Acquisition of cell polarization is essential for the performance of crucial functions, like a successful secretion and appropriate cell signaling in many tissues, and it depends on the correct functioning of polarity proteins, including the Crumbs complex. The CRB proteins, CRB1, CRB2 and CRB3, identified in mammals, are expressed in epithelial-de...

Apicobasal polarity is essential for epithelial cell function, and it is determined by the expression of three polarity protein complexes: Scribble, Par and Crumbs. Both the Par and the Crumbs complex, promote apical membrane identity. Crumbs complex is composed by PALS1, PATJ and the Crumbs homologs (CRB) proteins. We have recently described the e...

Dry eye disease is one of the most frequent pathological events that take place in the course of the graft versus host disease (GVHD), and is the main cause of deterioration in quality of life for patients. Thus, demonstration of dry eye signs in murine models of oGVHD is crucial for the validation of these models for the study of the disease. Give...

The mammalian central nervous system (CNS) is unable to regenerate. In contrast, the CNS of fish, including the visual system, is able to regenerate after damage. Moreover, the fish visual system grows continuously throughout the life of the animal, and it is therefore an excellent model to analyze processes of myelination and re-myelination after...

Myelinated axons in the retina. MBP+ axons (arrows) show a loose appearance in the retina when compared to ONH. Zn8+ growing axons (arrow heads) do not overlap with MBP+ axons. (TIF)

Clinical trials have assessed the use of human bone marrow stromal cells (hBMSCs) for the treatment of immune related disorders such as graft-versus-host disease (GVHD). In the current study we show that GFP+ transduced hBMSCs generated from bone marrow, migrate and differentiate into corneal tissue after subconjunctival injection in mice. Interest...

The CRB proteins CRB1, CRB2 and CRB3 are members of the cell polarity complex Crumbs in mammals that together with Scribble and Par complexes stablish the polarity of a variety of cell types. Although many members of the Crumbs complex proteins are expressed in the retinal pigment epithelium (RPE), and even though the mRNA of CRB2 has been detected...

During recent years a key role as morphogen has been postulated for the neurotransmitter acetylcholine in the developing Central Nervous System. Acetylcholine released from growing axons regulates growth, differentiation and plasticity. The acetylcholine distribution is frequently defined by acetylcholinesterase and choline acetyltransferase expres...

Purpose: Dry eye is a disease that results in tear film instability with potential damage to the ocular surface and is accompanied by an increased osmolarity of the tear film. One of the possible causes of dry eye is inflammation, as occurs in patients with graft versus host disease (GVHD). The purpose of the present study was to develop a new mode...

Retinoic acid (RA) is an important morphogen involved in retinal development. Perturbations in its levels cause retinal malformations such as microphthalmia. However, the cellular changes in the retina that lead to this phenotype are little known. We have used the zebrafish to analyse the effects of systemic high RA levels on retinogenesis. For thi...

CRB3 (Crumbs homologue 3), a member of the CRB protein family (homologous to the Drosophila Crumbs), is expressed in different epithelium-derived cell types in mammals, where it seems to be involved in regulating the establishment and stability of tight junctions and in ciliogenesis. This protein has been also detected in the retina, but little is...

Peptide competition assay for CRB3 and double immunolabeling CRB3/MPP4 and CRB3/rhodopsin. A–B, peptide competition assay for CRB3 where all the labeling disappears in retina cryosections at P0 (A) and at P4 (B). C–D, Immunofluorescence (C) and peptide competition assay (D) for the anti-MPP4 antibody in the adult mouse retina. C, MPP4 is present in...

CRB3 in the Müller glial cells. Double immunofluorescence for CRB3 (green) and CRALBP (red). A–C, CRALBP partially colocalizes with CRB3 in the Müller cells at the level of the OLM (arrowheads). D–F, CRALBP and CRB3 do not colocalize in any of the retinal plexiform layers, and the Müller cells processes seem to surround the CRB3 positive profiles (...

The transcription factor Pax2 actively participates in the development of the vertebrate visual system. In adults, Pax2 expression persists in a subpopulation of Müller cells and/or astrocytes in the retina and optic nerve head (ONH), although its function remains elusive. In a previous work we showed that the pax2 gene expression is modified and t...

The Pax2 transcription factor plays a crucial role in axon-guidance and astrocyte differentiation in the optic nerve head (ONH) during vertebrate visual system development. However, little is known about its function during regeneration. The fish visual system is in continuous growth and can regenerate. Müller cells and astrocytes of the retina and...

In this study we made intracellular injections of Lucifer Yellow fluoro c h rome into macro g l i a l cells, astrocytes and oligodendrocytes of the fixed optic nerve of tench (Tinca tinca ). From their three-dimensional morphology, we identified oligodendrocytes and at least four different types of astrocytes, both in the central zones of the nerve...

In this work, we used the pcd (Purkinje cell degeneration) mutant mouse with a slow temporal progression of photoreceptor degeneration in order to analyze the structural and functional modifications in the neuronal populations of the outer and inner retina. Retinal immunocytochemistry and functional electroretinography were performed on the pcd/pcd...

Embryonic exposure to ethanol leads to malformations such as cyclopia. Cyclopic embryos present fused eyes and lack of the ventral specification of the brain, with physiological and morphological defects in the visual system, which provides a useful model for teratology and neurotoxicity assessments. We analysed the differentiation of the visual ar...

During visual system morphogenesis, several cell populations arise at different time points correlating with the expression of specific molecular markers We have analysed the distribution pattern of three molecular markers (zn-1, calretinin and glial fibrillary acidic protein) which are involved in the development of zebrafish retina and optic tect...

The transcription factor Prox1 acts in rodent retinogenesis, at least in promoting cell cycle withdrawal and horizontal cell production. In the mature retina, this protein is detected at the inner nuclear layer of all vertebrate groups. We have made a neurochemical characterisation of Prox1(+) cell types in two different vertebrate groups: mammals...

We identified a new duplicated Dab1 gene (drDab1b) spanning around 25kb of genomic DNA in zebrafish. Located in zebrafish chromosome 2, it is composed of 11 encoding exons and shows high sequence similarity to other Dab1 genes, including drDab1a, a zebrafish Dab1 gene previously characterised. drDab1b encodes by alternative splicing at least five d...

Pax2 is a well known transcription factor which participates in optic nerve development. It assures the correct arrival and package of the newly formed retinal axons and the adequate differentiation of the newly formed glial cells. Pax2 protein expression is continuous throughout adult life in the goldfish optic nerve. We have found two populations...

We analyzed the modifications of the retinal neurons in a heterozygous mutant small eye mouse, the Sey(Dey). This mouse presents a mutation in chromosome 2 which affects the gene Pax6 and other nearby genes, such as the Wt1 gene and the gene of the Reticulocalbin. The eyes of these animals do not have lenses and their retinas present important morp...

The pattern of expression of the growth hormone (GH) gene was studied during the early development of gilthead sea bream ( Sparus aurata). The GH transcript was detected from the 2nd day of the larval stage onwards. In the next stages the expression level fluctuated, possibly due to different regulatory factors. The distribution of GH mRNA studied...

The patterns of expression of the somatolactin 1 and 2 (SL1 and SL2) transcripts were studied during the early development of the gilthead sea bream (Sparus aurata). Gene expression of SL1 and SL2 were detected in embryos and in larvae, although both transcripts presented different levels of expression. The SL1 transcripts in contrast to the SL2 tr...

We have analyzed the modifications in the tench (Tinca tinca) retina after the complete cryo-elimination of the proliferative growing zone (PGZ), which participates in the continuous growth of the retina throughout the life of the fish. By using immunohistochemistry and electron microscopy we demonstrated that, after the lesion, degenerative and re...

The expression of mRNAs of the two types of somatolactin which have been found, up to the present, in the pituitary of gilthead sea bream (Sparus aurata) have been analyzed by in situ hybridization (ISH). The method of non-radioactive ISH, which we optimize in this study, uses oligonucleotides labelled in the 5' end with biotin or dioxygenin as pro...

This study demonstrates the peculiarities of the glial organization of the optic nerve head (ONH) of a fish, the tench (Tinca tinca), by using immunohistochemistry and electron microscopy. We employed antibodies specific for the macroglial cells: glutamine synthetase (GS), glial fibrillary acidic protein (GFAP), and S100. We also used the N518 anti...

After a complete optic nerve section (ONS), retinal neurons may display retrograde transneuronal modifications in synaptic structure and function related to the retinal disconnection from the brain. The molecular and physiological basis of these changes is not yet fully understood. Immunoreactivity for calbindin was used to specifically immunolabel...

Although there is a lot information in the literature about genome size in fish, a high variability among data for the same species is reported, being mainly related to methodological aspects. Flow cytometry-based fluorescence measurements of intercalating dyes is the most attractive approach due to its precision, objectivity, high speed, and relat...

In the present study, a descriptive and quantitative analysis of all the proliferating cell populations present in the normal adult retina of three cyprinid species (goldfish, zebrafish, and tench) is reported. Evaluation of cell proliferation was performed in proliferating cell nuclear antigen (PCNA)-labeled tissue sections as well as in single-ce...

Localization of mRNA of two types of somatolactin, growth hormone, and prolactin in the pituitary of gilthead seabream Sparus auratus L., 1758 The mRNA expression of two types of somatolactin (SL1 and SL2), growth hormone (GH), and prolactin (PRL) were analysed by in situ non-radioactive hybridization (ISH) in the pituitary of gilthead sea bream Sp...

The mRNA expression of two types of somatolactin (SL1 and SL2), growth hormone (GH), and prolactin (PRL) were analysed by in situ non-radioactive hybridization (ISH) in the pituitary of gilthead sea bream Sparus auratus L., 1758. The different SL probes used, 5' end labelled with biotin or dioxygenin, showed a strong signal, mainly in the pars inte...

We quantitatively evaluate the changes of the proliferative cell populations in the adult tench retinas maintained at 6°C and 20°C by both PCNA antigen detection and flow cytometry-based DNA measurements. Both the overall percentage of S-phase cells in the whole retinas and the number of PCNA-positive cells in each of the retinal layers were signif...

In the present work we show that during the degenerative process occurring after the cryo-elimination of the tench peripheral growing zone many non-neuronal cell types in addition to the resident microglial cells, appear within the affected areas. Some of them are normally found in the retina, such as the retinal pigmented epithelium cells and othe...

The present study is a morphological and quantitative analysis of protein kinase C-like immunoreactive (PKC-L ir) bipolar cells in the retinas of five different vertebrate species (chicken, tench, zebrafish, goldfish and rat). The morphology of PKC-L-ir bipolar cell axon terminals in fish differs significantly from those of chicken and rat retinas....

We have analyzed the immunolabeling with the antibody RT97, a good marker for ganglion cell axons in several species, in the normal and regenerating visual pathways of teleosts. We have demonstrated that RT97 antibody recognizes several proteins in the tench visual system tissues (105, 115, 160, 200, 325 and 335 kDa approximately). By using immunop...

Using monoclonal antibodies against parvalbumin (PV) and calbindin (CB), and a polyclonal antiserum against calretinin (CR), the expression patterns of these proteins in the retina of the tench and rainbow trout were studied at light microscopic level in in toto preparations and radial sections. Parvalbumin was present in subpopulations of small am...

Morphological evidence of a temporal parallelism between the appearance of the α isoform of protein kinase C (PKC) and some processes such as synaptogenesis in the plexiform layers of the chicken retina is offered. Immunostaining experiments were performed throughout embryonic, young and adult chicken life. The results help to understand the develo...

Histochemistry for nucleoside diphosphatase was used to study the microglial cells in the adult tench retina. An abundant population of microglial cells was located in the vascular membrane, nerve fibre layer, inner and outer plexiform layers and scattered cells were observed in the inner nuclear layer. Rounded and amoeboid cells could be seen clos...

We studied the glial response after inducing a lesion in the zone of the peripheral retina of tench, where there is proliferative neuroepithelium. In the retina and optic nerve, the microglial response was analysed with tomato lectin and the macroglial response with antibodies against GFAP and S-100. In lesioned retinas, there was a temporal–spatia...

By cryolesion of the scleral-corneal perimeter of the eye of a teleost we eliminated both the marginal growth ring and the immature portions adjacent to the neural retina. Electron microscopy revealed necrotic destruction of the whole of the germinative zone and a large number of apoptotic cells in neighbouring zones. In the degenerative processes...

Different parts of the tench optic nerve, the intraocular and intraorbital segments, the chiasm, and the post-chiasmatic segment, were studied using light and electron microscopy. From the head of the optic nerve, a zone of continuous growth constituted by the younger non-myelinated ganglion axons can be differentiated from a mature zone where almo...

The location of several diencephalic and mesencephalic structures in the teleost fish, Tinca tinca, which have not been described previously, was made possible by injecting Fluoro-Gold, as an anterograde and retrograde tracer, into the optic nerve. In the pretectal area, we found the tractus opticus accessorius and the nucleus opticus dorsolaterali...

Triton X-100 is used in immunohistochemistry to make tissue permeable, to present certain antigens to antisera, and to prevent certain nonspecific interactions. This detergent is routinely dissolved in buffers at concentrations of 0.01-0.2%. Using high concentrations of Triton X-100 (0.2-2%) and anti-immunoglobulins G (anti-IgGs), labeling of myeli...

Continual growth and regenerative capacity are characteristics of the Central Nervous System (CNS) of some teleosts, and both particularities are especially notable in the visual system. Within this system, the optic nerve is an ideal structure for the study of the glia of these animals and of their involvement in the processes of development and r...

The intensity of the immunohistochemical reaction (IIR) against the alpha species of protein kinase C (PKC) was quantified in the rod bipolar cells (RBC) of the goldfish retina using of image analysis. Retinae incubated in control Ringer solution showed similar IIR in both the soma and the axon terminal (IIR-ratio approximately 1). Activation of PK...

Glial cells in the normal and regenerating visual pathways of Tinca tinca (Cyprinid, Teleost) were studied by labelling with anti-S-100 antibody. In normal fish, S-100-positive bipolar cells were found in the optic nerve, optic tract, and in the diencephalic visual pathways. After crushing the left optic nerve, the distribution and the number of S-...

The distribution of S100 immunoreactivity within the normal and regenerating retina and optic nerve head of the teleost Tinca tinca L. has been investigated using the avidin-biotin complex (ABC) method and a polyclonal antibody against S100. Astrocytes and Müller cells were labeled with this antibody. This represents the first description of astroc...

The purpose of the present study was to elucidate the physiological mechanisms that determine the activation of protein kinase C (PKC) in rod bipolar cells (RBC) of mouse and goldfish. The localization of PKC in RBC was examined using immunoreactivity (IR) against the alpha species of the enzyme. After incubating the whole retina or dissociated cel...

Purpose: Bipolar cell axon terminals receive direct synaptic inputs from amacrine cell processes in the vertebrate retina, suggesting a possible pathway for modulation of transmitter release at this level. Rod bipolar cells (RBCs) are immunoreactive (IR) to the α species of protein kinase C (PKC). The effect of several PKC activators and/or amacrin...

We have studied the microglial cells in the normal and regenerating visual pathways of Tinca tinca (Cyprinid, Teleost) by using the lectin from Lycopersicum esculentum (tomato), which, in our case, has been demonstrated as a specific marker for teleost microglia. In the normal fish, there are tomato lectin positive microglial cells in the retina, o...

The distribution of parvalbumin immunoreactivity in the telencephalic hemispheres of the tench (Tinca tinca L.) was studied using a monoclonal antibody and the avidin-biotin immunoperoxidase method. A wide distribution of immunoreactive structures was found in both dorsal and ventral areas of the telencephalic hemispheres. Normally, the parvalbumin...

The appearance of calbindin D-28K, a calcium-binding protein, during development of the trout retina was studied by immunohistochemistry. The first calbindin immunoreactive cells appear in the inner nuclear layer at the equator of the embryonic retina at the stage 227 degrees C (around embryonic day 15). Just before hatching, stage 440 degrees C (a...

The distribution of neurokinin A-like immunoreactive cell bodies and fibers in the diencephalon of the cat was studied using an indirect immunoperoxidase technique. A high or moderate density of immunoreactive neurons was observed in the nuclei habenularis lateralis, medialis dorsalis, parafascicularis, hypothalamus posterior, area hypothalamica do...

This report provides an atlas of the tench brain in the transverse plane. The brain atlas is based on paraffin-embedded brains which are serially sectioned and stained with cresyl violet. It consists of drawings and photographs at several levels throughout the main divisions of the tench brain. The brain of the tench shows important variations in t...