Publicaciones por año
Publicaciones del Departamento de Histología y Embriología
A rapid method for fibronectin purification on nitrocellulose membranes suitable for tissue culture
J Biochem Biophys Methods 2004 May 31;59(2):139-43
Silvia Chifflet 1, Carmen Bolatto 2, Susana Tolosa 1
1 Departamento de Bioquı́mica, Facultad de Medicina, Universidad de la República, Gral. Flores 2125, 11800 Montevideo, Uruguay 2 Departamento de Histologı́a y Embriologı́a, Facultad de Medicina, Universidad de la República, Gral. Flores 2125, 11800 Montevideo, Uruguay
DOI: 10.1016/j.jbbm.2003.12.004
PMID: 15163525
Pubmed: https://pubmed.ncbi.nlm.nih.gov/15163525
Texto completo: https://linkinghub.elsevier.com/retrieve/pii/S0165022X03002161
Abstract:
We have developed a simple method for plasma fibronectin purification based on the well-known gelatin binding property of fibronectin. In this procedure we immobilize the melted gelatin to nitrocellulose membranes; these are then used to affinity-purify the fibronectin from the plasma sample. The fibronectin is eluted from the membrane by treatment with 8 M urea. The procedure described here gives a yield of up to 60% (from presumed fibronectin concentration) and the fibronectin obtained is homogeneous in SDS-PAGE and biologically active, as assessed by a cell migration assay. The method is rapid, simple, inexpensive, does not require the use of chromatographic equipment and is suitable for tissue culture applications.
Induction of motor neuron apoptosis by free 3-nitro-L-tyrosine
J Neurochem 2004 May;89(3):602-12
Hugo Peluffo 1, John J Shacka 7, Karina Ricart 7, C Gaston Bisig 3, Laura Martìnez-Palma 1, Otto Pritsch 2, Andrès Kamaid 1, Jason P Eiserich 4, John P Crow 5, Luis Barbeito 6, Alvaro G Estèvez 7
Departamentos de 1 Histologia y Embriología and 2 Bioquimica, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay 3 Departamento de Química Biologica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba , Córdoba, Argentina 4 Department of Internal Medicine and Human Physiology, University of California at Davis, Davis, California, USA 5 Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, LittleRock, Arkansas, USA 6 División Neurobiología Celular y Molecular, Instituto de Investigaciones Biológicas Clemente Estable, Montevideo, Uruguay 7 Department of Physiology and Biophysics, and 8 Department of Neurobiology and Center for Free Radical Biology, The University of Alabama at Birmingham, Birmingham, Alabama, USA
DOI: 10.1046/j.1471-4159.2004.02363.x
PMID: 15086517
Pubmed: https://pubmed.ncbi.nlm.nih.gov/15086517
Texto completo: https://onlinelibrary.wiley.com/doi/10.1046/j.1471-4159.2004.02363.x
Abstract:
Peroxynitrite-dependent tyrosine nitration has been postulated to be involved in motor neuron degeneration in amyotrophic lateral sclerosis (ALS). Evidence supporting this supposition includes the appearance of both free and protein-linked 3-nitro-l-tyrosine (nitrotyrosine) in both sporadic and familial ALS, as well as of increased free nitrotyrosine levels in the spinal cord of transgenic mice expressing ALS-linked superoxide dismutase mutants at symptom onset. Here we demonstrate that incubation with clinically relevant concentrations of nitrotyrosine induced apoptosis in motor neurons cultured with trophic factors. Nitrotyrosine was bound to proteins, but it was not incorporated into alpha-tubulin, as previously demonstrated for other cell types. Neither inhibition of nitric oxide production nor scavenging of superoxide and peroxynitrite prevented increases in cell nitrotyrosine immunoreactivity or motor neuron death, suggesting that these effects are not due to the endogenous formation of reactive nitrogen species. In contrast, some populations of astrocytes incorporated nitrotyrosine into alpha-tubulin, but free nitrotyrosine had no effect on the viability and phenotype of astrocytes in culture, as evaluated by glial fibrillary acidic protein immunoreactivity, cell growth and morphology. Co-culture of motor neurons on astrocyte monolayers delayed, but did not prevent, nitrotyrosine-induced motor neuron death. These results suggest that free nitrotyrosine may play a role in the induction of motor neuron apoptosis in ALS.
Environment temperature affects cell proliferation in the spinal cord and brain of juvenile turtles
J Exp Biol 2003 Sep;206(Pt 17):3085-93
Milka Radmilovich 1, Anabel Fernández 2 3, Omar Trujillo-Cenóz 2 3
1 Departamento de Histología y Embriología, Facultad de Medicina, Montevideo, Uruguay ZC 11800 2 Laboratorio de Neuroanatomía Comparada, Instituto de Investigaciones Biológicas Clemente Estable, Avda. Italia 3318,Montevideo, Uruguay ZC 11600 3 Unidad Asociada a la Facultad de Ciencias, Montevideo, Uruguay ZC 11600
DOI: 10.1242/jeb.00515
PMID: 12878675
Pubmed: https://pubmed.ncbi.nlm.nih.gov/12878675
Texto completo: https://journals.biologists.com/jeb/article-lookup/doi/10.1242/jeb.00515
Abstract:
The spinal cords and brains--comprising dorsal cortex (DC), medial cortex (MC) and diencephalon (Dien)--of juvenile turtles acclimated to warm temperature [27-30 degrees C; warm-acclimated turtles (WATs)] revealed higher density values of bromodeoxyuridine-labeled cells (BrdU-LCs) than those acclimated to a cooler environment [5-14 degrees C; cold-acclimated turtles (CATs)]. Both populations were under the influence of the seasonal daily light-dark rhythms. Pronounced differences between WATs and CATs (independent t-test; confidence level, P<0.01) were found in the central area of the spinal gray matter and in the ependymal epithelium lining the brain ventricles. Forebrain regions (DC, MC and Dien) also revealed significant differences between WATs and CATs (independent t-test; confidence level, P<0.01-0.05). Unexplored biological clocks that may be affecting cell proliferation were equalized by performing paired experiments involving one WAT and one CAT. Both animals were injected on the same day at the same time and both were sacrificed 24 h later. These experiments confirmed that a warm environment increased cell proliferation in the CNS of turtles. Double- and triple-labeling experiments involving anti-BrdU antibody together with anti-glial protein antibodies revealed that temperature modulates not only cell populations expressing glial markers but also other cells that do not express them. As expected, in the case of short post-injection (BrdU) surviving time points, no cells were found colabeling for BrdU and NeuN (neuronal marker). The probable direct effect of temperature on the cell division rate should be analyzed together with potential indirect effects involving increased motor activity and increased food intake. The fate of the increased BrdU-LCs (death, permanence as progenitor cells or differentiation following neuronal or glial lines) remains a matter for further investigation. Results are discussed in the light of current opinions concerned with post-natal neurogenesis in vertebrates.
Synaptic activity modifies the levels of Dorsal and Cactus at the neuromuscular junction of Drosophila
J Neurobiol 2003 Feb 15;54(3):525-36
Carmen Bolatto 1 , Silvia Chifflet 2 , Aram Megighian 3 , Rafael Cantera 4
1 Departamento de Histología y Embriología, Facultad de Medicina, Universidad de la República, Uruguay. 2 Departamento de Bioquímica, Facultad de Medicina, Universidad de la República, Uruguay 3 Aram Megighian, Department of Human Anatomy and Physiology, University of Padova, Italy 4 Zoology Department, Svante Arrheniusväg 14, Stockholm University, Stockholm, Sweden
DOI: 10.1002/neu.10179
PMID: 12532402
Pubmed: https://pubmed.ncbi.nlm.nih.gov/12532402
Texto completo: https://doi.org/10.1002/neu.10179
Abstract:
The Drosophila Rel transcription factor Dorsal and its inhibitor Cactus participate in a signal transduction pathway involved in several biologic processes, including embryonic pattern formation, immunity, and muscle development. In contrast with embryonic muscle, where Dorsal is reportedly absent, this protein and Cactus accumulates in the neuromuscular junctions in the muscle of both larvae and adults. The phenotype of homozygous dorsal mutant larvae suggested that Dorsal and Cactus maybe necessary for normal function and maintenance of the neuromuscular system. Here we investigate if these proteins can respond to synaptic activity. Using larval body wall preparations and antibodies specific for Dorsal or Cactus we show that the amount of these proteins at the neuromuscular junction is substantially decreased after electrical stimulation of the nerves or incubation in glutamate, the principal transmitter in this type of synapse. The specificity of the response was tested with a glutamate receptor antagonist (argiotoxin 636). Because the effect can be reproduced using a calcium ionophore (ionomycin treatment) as well as blocked by the inhibition of the muscle ryanodine receptor (tetracaine treatment), the involvement of calcium in this process seems likely. We also observed that the inhibition of the calcium dependent protein phosphatase calcineurin prevents the effect of glutamate on the fluorescence for Dorsal and Cactus, suggesting its participation in a signal transduction cascade that may activate Dorsal in the muscle independently of Toll. Our results are consistent with a novel function of the Rel factor Dorsal in a molecular pathway turned on by neural activity and/or contractile activity.
Involvement of nitric oxide on kainate-induced toxicity in oligodendrocyte precursors
Neurotox Res 2003 2003;5(6):399-406
Laura Martinez-Palma 1, Mariana Pehar 2, Patricia Cassina 1, Hugo Peluffo 1, Raquel Castellanos 1, Gabriel Anesetti 1, Joseph S Beckman3 , Luis Barbeito 2
1 Departamento de Histologia, Facultad de Veterinaria y Facultad de Medicina, Universidad de la República, 11600, Montevideo, Uruguay Laura Martinez-Palma, Patricia Cassina, Hugo Peluffo, Raquel Castellanos & Gabriel Anesetti 2 Departamento de Neurobiología Celular, Instituto de Investigaciones Biológicas Clemente Estable, Av. Italia 3318, 11600, Montevideo, Uruguay Mariana Pehar & Luis Barbeito 3 Linus Pauling Institute, Department of Biochemistry and Biophysics, Oregon State University, 97331, Corvallis, OR, USA Joseph S. Beckman
DOI: 10.1007/BF03033168
PMID: 14715442
Pubmed: https://pubmed.ncbi.nlm.nih.gov/14715442
Texto completo: https://dx.doi.org/10.1007/BF03033168
Abstract:
The vulnerability of oligodendrocytes to excitatory amino acids may account for the pathology of white matter occurring following hypoxia/ischemia or autoimmune attack. Here, we examined the vulnerability of immature oligodendrocytes (positively labeled by galactocerobroside-C and not expressing myelin basic protein) from neonatal rat spinal cord to kainate, an agonist of excitatory amino acid receptors that induces long-lasting inward currents in immature oligodendrocytes. In particular, we studied whether kainate toxicity was linked to the endogenous production of nitric oxide. We found cultured oligodendrocytes to be highly sensitive to 24-48 h exposure to 0.5-1 mM kainate. The toxin induced striking morphological changes in oligodendrocytes, characterized by the disruption of the process network around the cell body and the growth of one or two long, thick and non-branched processes. A longer exposure to kainate resulted in massive death of oligodendrocytes, which was prevented by 6,7, dinitroquinoxaline-2,3-dione (DNQX) (30 micro M), the antagonist of AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic/kainate receptors. Remarkably, we found that those oligodendrocytes displaying bipolar morphology following kainate exposure, also expressed the inducible form of nitric oxide synthase (iNOS) and nitrotyrosine immunoreactivity, suggesting that peroxynitrite could be formed by the reaction of nitric oxide with superoxide. Moreover, kainate toxicity was significantly prevented by addition of the NOS inhibitor nitro-L-arginine methyl ester (L-NAME), further suggesting that nitric oxide-derived oxidants contribute to excitotoxic mechanisms in immature oligodendrocytes.
Intrinsic neurons in the mammalian ovary
Microsc Res Tech 2002 Dec 15;59(6):484-9
Hortensia D'Albora 1 , Gabriel Anesetti 1 2, Paula Lombide 1 3, W Les Dees 4, Sergio R Ojeda 5
1 Departamento de Histología y Embriología, Facultad de Medicina, CP 11800 Montevideo, Uruguay. hdalbora@fmed.edu.uy 2 Area de Histología y Embriología, Facultad de Veterinaria, CP 11600 Montevideo, Uruguay 3 Sección Biología Celular, Facultad de Ciencias, CP 11400 Montevideo, Uruguay 4 Department of Anatomy, College of Veterinary Medicine, Texas A&M University, College Station, TX 77843 5 Division of Neuroscience, Oregon Regional Primate Research Center, Oregon Health Sciences University, OR 97006
DOI: 10.1002/jemt.10231
PMID: 12467023
Pubmed: https://pubmed.ncbi.nlm.nih.gov/12467023
Texto completo: https://doi.org/10.1002/jemt.10231
Abstract:
Mammalian ovarian function is under endocrine and neural control. Although the extrinsic innervation of the ovary has been implicated in the control of both ovarian development and mature function, it is now clear that, from rats to humans, the ovary is endowed with a network of intrinsic neurons displaying diverse chemical phenotypes. This article describes the presence of these intrinsic neurons in the ovary of different mammalian species, and discusses the possible functions that they may have in the regulation of ovarian physiology.
The oestrogenized rat myometrium inhibits organotypic sympathetic reinnervation
Auton Neurosci 2002 Oct 31;101(1-2):13-22
M M Brauer a, R Chávez-Genaro a b, A Richeri a, L Viettro a, A I Frias c, G Burnstock d, T Cowen e
a Laboratorio de Biologı́a Celular, Instituto de Investigaciones Biológicas Clemente Estable, Avenida Italia 3318, Montevideo 11600, Uruguay b Departamento de Histologı́a, Facultad de Medicina, Universidad de la Repūblica, Montevideo, Uruguay c Departamento de Biologı́a Animal y Humana, Facultad de Biologı́a, Universidad de La Habana, Havana, Cuba d Autonomic Neuroscience Institute, Royal Free and University College Medical School, London, UK e Department of Anatomy and Developmental Biology, University College London, Royal Free Campus, London NW3 2PF, UK
DOI: 10.1016/s1566-0702(02)00173-x
PMID: 12462355
Pubmed: https://pubmed.ncbi.nlm.nih.gov/12462355
Texto completo: https://www.autonomicneuroscience.com/article/S1566-0702(02)00173-X/abstract
Abstract:
Chronic administration of oestrogen to rats during the infantile/prepubertal period provokes, at 28 days of age, complete loss of noradrenaline-labelled intrauterine sympathetic nerves. It is not known whether oestrogen inhibits the growth or causes the degeneration of developing uterine sympathetic nerves, or whether the uterus recovers its innervation following cessation of infantile/prepubertal oestrogen treatment. In the present study, we analysed the time-course of the effects of oestrogen on the development of uterine sympathetic nerves in the rat, using histochemical methods. In addition, the pattern of sympathetic reinnervation of the uterus of intact and ovariectomised females was assessed 3 and 6 months after cessation of chronic oestrogen treatment. The ability of sympathetic nerves to reinnervate the oestrogenized uterine tissue was assessed in intraocular transplants of uterine myometrium into ovariectomised host rats. Early exposure to oestrogen did not inhibit the approach of sympathetic nerves to the uterus, but prevented the normal growth and maturation of intrauterine sympathetic fibres and abolished the innervation that reached the organ before initiation of treatment. Three or six months following cessation of oestrogen treatment, most of the sympathetic nerves were restricted to the mesometrium and mesometrial entrance, whereas intrauterine innervation remained persistently depressed as a consequence of a sustained oestrous-like state provoked by ovarian dysfunction (polycystic ovary). An organotypic regrowth of uterine sympathetic nerves was observed in ovariectomised infantile/prepubertal oestrogen-treated animals. After 5 weeks in oculo, the innervation of oestrogenized myometrial transplants was reduced by 50%, and substantial changes in the pattern of reinnervation were observed. In control transplants, 86% of the nerves were terminal varicose myometrial and perivascular nerve fibres, whereas 14% were preterminal nerve bundles. In oestrogenized myometrial transplants, 83% of the noradrenaline-labelled intercepting nerves were enlarged preterminal bundles and only 17% were terminal fibres. These results indicate that the oestrogenized myometrium is unattractive for sympathetic nerves and inhibits organotypic sympathetic reinnervation.
Peroxynitrite-induced cytotoxicity in cultured astrocytes is associated with morphological changes and increased nitrotyrosine immunoreactivity
Neurotox Res 2002 Mar;4(2):87-93
Mariana Pehar 1 2, Laura Martínez-Palma 2, Hugo Peluffo 2, Andrés Kamaid 2, Patricia Cassina 2, Luis Barbeito 1
1 Neurobiología Celular y Molecular, Instituto de Investigaciones Biológicas Clemente Estable, Montevideo, Uruguay 2 Departamento Histología y Embriología, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay
DOI: 10.1080/10298420290015818
PMID: 12829407
Pubmed: https://pubmed.ncbi.nlm.nih.gov/12829407
Texto completo: https://link.springer.com/article/10.1080/10298420290015818
Abstract:
We have established a cell culture model of spinal cord astrocytes to study the cytotoxicity of peroxynitrite. Nitric oxide (NO) has been implicated as a key contributor to neurotoxicity. NO reacts with superoxide to generate peroxynitrite, a strong oxidant and nitrating agent with deleterious cytotoxic and pro-apoptotic effects. Peroxynitrite and nitrotyrosine are formed in damaged motor neurons in amyotrophic lateral sclerosis (ALS), which are surrounded by reactive astrocytes. To determine the effects of extracellular addition of peroxynitrite, purified astrocyte monolayers prepared from neonatal rat spinal cords were exposed to peroxynitrite (0.25-0.75 mM) for 5 min and further incubated in culture medium for 24-72h. Peroxynitrite exposure did not result in apparent cell loss or damage of the monolayer. However, a substantial number of cells adopted reactive features, with long processes displaying intense immunoreactivity to glial fibrillary acidic protein (GFAP). Western blot analysis performed 24h after peroxynitrite treatment showed that GFAP levels were not modified by the oxidant. There were no changes in cell viability parameters in astrocyte cultures after peroxyintrite, indicating that astrocytes are more resistant to the oxidant than other cell types. Peroxynitrite reacts with protein-bound tyrosine residues to form nitrotyrosine. We observed a modest to strong nitrotyrosine immunoreactivity in astrocytes 24h following peroxynitrite exposure. There was a remarkable association between nitrotyrosine and high-intensity GFAP immunoreactivity in astrocytes bearing long processes. These results suggest that peroxynitrite induces a characteristic long-lasting reactive astrocytic phenotype and provide new insight into understanding the origin of reactive astrocytes occurring in ALS.
Peroxynitrite triggers a phenotypic transformation in spinal cord astrocytes that induces motor neuron apoptosis
J Neurosci Res 2002 Jan 1;67(1):21-9
Patricia Cassina 1 , Hugo Peluffo 1, Mariana Pehar 1 2, Laura Martinez-Palma 1 3, Andrés Ressia 1, Joseph S Beckman 4, Alvaro G Estévez 5, Luis Barbeito 2
1 Departamento de Histología y Embriología, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay. 2 Departamento de Neurobiología Celular y Molecular, Instituto de Investigaciones Biológicas Clemente Estable, Montevideo, Uruguay 3 Facultad de Veterinaria, Universidad de la República, Montevideo, Uruguay 4 Linus Pauling Institute, Department of Biochemistry and Biophysics, Oregon State University,Corvallis, Oregon 5 Department of Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, Alabama
DOI: 10.1002/jnr.10107
PMID: 11754077
Pubmed: https://pubmed.ncbi.nlm.nih.gov/11754077
Texto completo: https://doi.org/10.1002/jnr.10107
Abstract:
Oxidative stress mediated by nitric oxide (NO) and its toxic metabolite peroxynitrite has previously been associated with motor neuron degeneration in amyotrophic lateral sclerosis (ALS). Degenerating spinal motor neurons in familial and sporadic ALS are typically surrounded by reactive astrocytes expressing the inducible form of NO synthase (iNOS), suggesting that astroglia may have a pathogenic role in ALS. We report here that a brief exposure of spinal cord astrocyte monolayers to peroxynitrite (0.25-1 mM) provoked long-lasting reactive morphological changes characterized by process-bearing cells displaying intense glial fibrillary acidic protein and iNOS immunoreactivity. Furthermore, peroxynitrite caused astrocytes to promote apoptosis of embryonic motor neurons subsequently plated on the monolayers. Neuronal death occurred within 24 hr after plating, as evidenced by the presence of degenerating motor neurons positively stained for activated caspase-3 and nitrotyrosine. Motor neuron death was largely prevented by NOS inhibitors and peroxynitrite scavengers but not by trophic factors that otherwise will support motor neuron survival in the absence of astrocytes. The bacterial lipopolysaccharide, a well-known inflammatory stimulus that induces iNOS expression in astrocytes, provoked the same effects on astrocytes as peroxynitrite. Thus, spinal cord astrocytes respond to extracellular peroxynitrite by adopting a phenotype that is cytotoxic to motor neurons through peroxynitrite-dependent mechanisms.
Neural retina of chick embryo in organ culture: effects of blockade of growth factors by suramin
Cell Tissue Res 2001 Jun;304(3):323-31
A Cirillo 1 , S Chifflet 2 , B Villar 1
1 Departmento de Histología y Embriología, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay. acirillo@fmed.edu.uy 2 Departamento de Bioquímica. Facultad de Medicina, Universidad de la República, Montevideo, Uruguay
DOI: 10.1007/s004410100355
PMID: 11456408
Pubmed: https://pubmed.ncbi.nlm.nih.gov/11456408
Texto completo: https://dx.doi.org/10.1007/s004410100355
Abstract:
The neural retina is a highly organized organ whose final histoarchitecture depends on the presence of diverse growth factors and on their interactions with extracellular matrix components. However, the role of growth factors on retinal development is not fully understood. Suramin has been shown to produce diverse cellular effects via the simultaneous block of the action of several growth factors. We have therefore studied the effects of suramin on organotypic culture of chick embryo neural retina in order to gain further insights into the participation of growth factors in neural retinal development. Neural retina was incubated for 24 h with suramin at 50-200 microM and then processed to determine cell proliferation, nuclear morphology, and actin distribution. Suramin provoked extensive morphological changes revealed by a decrease in BrdU incorporation, alterations in cellular organization, and disruption of the outer limiting membrane, with the emergence of cellular elements through it. All of these effects were dose-dependent and markedly attenuated by the simultaneous presence of suramin and fibroblast growth factor 2 (FGF-2) in the culture medium. These findings indicate that suramin induces pleiotropic effects on the histoarchitecture of the chicken neural retina in organ culture and suggest that FGF-2 is one of the biological modulators involved in the maintenance of the structural organization of the chicken neural retina.