Hernán Hurtado

Schwann cell-enriched cultures from adult human peripheral nerve: a technique combining short enzymatic dissociation and treatment with cytosine arabinoside (Ara-C)

Attempts to design the nerve cellular prostheses have focused on the production of autologous Schwann cells expanded in vitro as the essential component in the regeneration process of injured peripheral nerves. To obtain human Schwann cells of high quality we tested a short enzymatic dissociation protocol that optimized cellular viability levels. We also assessed patterns of bromodeoxyuridine (BrdU) incorporation in both Schwann cells and fibroblasts in the presence or absence of the antimitotic Ara-C, an enrichment option for adult human Schwann cell cultures. The Ara-C treated cultures showed a significantly higher Schwann cell percentage (95%), compared with that obtained in the absence of Ara-C (70%), indicating that this antimitotic acts to eliminate fibroblasts in each one of the applied pulses (four pulses). However, we have observed that the use of this antimitotic during prolonged periods of time produced a cumulative effect causing Schwann cell cytotoxicity. Therefore, we consider that our enzymatic dissociation technique and the application of only two pulses of Ara-C to the cultures are enough to achieve enrichment of adult human Schwann cells in culture.

Nerve growth factor and Neurotrophin-3 modulate the rabies infection of adult sensory neurons in primary cultures

With the aim of determining if the proportion of rabies virus (RV)-infected adult neurons from dorsal root ganglion are affected by in vitro treatment with different neurotrophins, experiments using Nerve Growth Factor (NGF), Brain Derived Neurotrophic Factor (BDNF) or Neurotrophin-3 (NT-3) as supplements for cells in culture were performed. Cultures treated with three different concentrations of each of the neurotrophins mentioned were infected with Challenge Virus Standard RV strain. An indirect immunoperoxidase technique was performed for the detection and counting of infected cells. NGF (2 ngml(-1) and 10 ngml(-1)) and NT-3 (1 ngml(-1) and 5 ngml(-1)) induced a significant reduction of infected neurons. None of the cultures treated with BDNF showed changes in the percentage of infected neurons. Likewise, the proportion of infected non-neuronal cells (Schwann cells and fibroblasts) was not altered by the treatment with neurotrophins. In addition, morphometric analysis of total and virus-immunoreactive neurons in culture were carried out, the neurotrophin treatment induced variations in the profile of neurons preferentially infected, since cell diameters in the infected cell population are different in the presence of NGF and NT-3. Data presented here could indicate a putative participation of neurotrophin receptor or biochemical modifications induced by neurotrophin treatment that affect the infection. The primary culture of dorsal root ganglion cells from adult mice is a very useful model for studying the basic phenomena of the RV-neuron interaction.

Partial inhibition of the in vitro infection of adult mouse dorsal root ganglion neurons by rabies virus using nicotinic antagonists.

The infection of target cells by rabies virus is effected through membrane receptors. Several authors have suggested that nicotinic receptors could be used by this virus, but no direct experimental evidence is available. In this study mouse dorsal root ganglia cells were treated with various nicotinic antagonists (dihydro-beta-eritroidine, mecamilamine, d-tubocurarin, hexametonium, alpha-bungarotoxin and erabutoxin). After incubation, the cultures were infected with rabies virus. Cells were fixed, and processed for immunodetection of rabies virus. Treatment with mecamilamine or d-tubocurarine reduced the percentage of infected neurons. None of the antagonists tested changed the percentage of infected non-neuronal cells.

Rabies virus infection of cultured adult mouse dorsal root ganglion neurons

An in vitro model of adult dorsal root ganglion neurons infection by rabies virus is described, Viral marked neurotropism is observed, and the percentage and the degree of infection of the neurons is higher than in non neuronal cells, even if neurons are the minority of the cells in the culture. The neuritic tree is also heavily infected by the virus.

Estudio Histológico del Tracto Digestivo del Neón Cardenal Paracheirodon axelrodi (Characidae)

Paracheirodon axelrodi es el pez ornamental que mas se exporta en Colombia. Sin embargo, se desconocen varios aspectos de su biologia basica. Se estudio la histologia del tracto digestivo. Se sacrificaron 15 ejemplares (MS 222, 0,5 g/L), fijados en formaldehido al 4 % y decalcificados con acido nitrico 7 %. Se siguio el procedimiento para H&E. El tracto digestivo de P. axelrodi presento cuatro capas constitutivas: mucosa, submucosa, muscular y serosa. La mucosa de la boca presento un epitelio escamoso estratificado no queratinizado con celulas caliciformes y sin botones gustativos, una submucosa de tejido conectivo laxo y una capa de musculo estriado esqueletico. Los dientes son conicos y abundantes. La capa serosa a lo largo del tracto digestivo es delgada compuesta de tejido conectivo laxo. La capa mucosa, submucosa y muscular del esofago posee la misma conformacion tisular de la boca, sin embargo, la capa muscular presenta dos orientaciones. La mucosa del estomago esta compuesta por epitelio cilindrico simple con glandulas gastricas, submucosa de tejido conectivo laxo y una capa de musculo liso en dos orientaciones. Las capas del intestino exhi-bieron una composicion tisular similar a la del estomago, sin glandulas gastricas. La mucosa presento celulas caliciformes especialmente hacia la region posterior y un aumento en la longitud de los pliegues intestinales. La conformacion tisular y morfologica del tracto digestivo de P. axelrodi lo ubica como un pez de habitos alimenticios carnivoros pero de pequenas presas.

Ultrastructural description of rabies virus infection in cultured sensory neurons

Primary cultures were made from adult mouse spinal ganglia for depicting an ultrastructural description of rabies virus (RABV) infection in adult mouse sensory neuron cultures; they were infected with rabies virus for 24, 36, and 48 h. The monolayers were processed for transmission electron microscopy and immunochemistry studies at the end of each period. As previously reported, sensory neurons showed great susceptibility to infection by RABV; however, in none of the periods evaluated were assembled virions observed in the cytoplasm or seen to be associated with the cytoplasmic membrane. Instead, fibril matrices of aggregated ribonucleoprotein were detected in the cytoplasm. When infected culture lysate were inoculated into normal animals via intra-cerebral route it was observed that these animals developed clinical symptoms characteristic of infection and transmission electron microscopy revealed assembled virions in the cerebral cortex and other areas of the brain. Sensory neurons infected in vitro by RABV produced a large amount of unassembled viral ribonucleoprotein. However, this intracellular material was able to produce infection and virions on being intra-cerebrally inoculated. It can thus be suggested that the lack of intracellular assembly in sensory neurons forms part of an efficient dissemination strategy.

Studying neurotrophin antiviral effect on rabies-infected dorsal root ganglia cultures

Neurotrophin (NT)-induced modulation of rabies virus adsorption, transcription, and replication were analyzed in adult mouse dorsal root ganglia cultures. Different types of nerve growth factor and NT-3 treatment were tested before infection (pretreatment), during infection (transtreatment) and after withdrawing the viral inoculum (post-treatment). NT pretreatment for 4 days prior to infection produced a significant increase in the quantity of virus adsorbed into cultures and a concomitant increase in genomic viral RNA as measured by real time polymerase chain reaction (PCR). NT pretreatment triggered increased expression of two rabies virus receptors (NCAM and p75NTR); however, no increase in rabies virus transcription and expression could be observed. By contrast, NT treatment during and after infection (trans- and post-treatment) induced a strong decrease in the quantity of viral nucleoprotein genomic and messenger nucleoprotein RNAs. These findings suggested that NT had an intrinsic inhibitory effect on rabies virus infection, which was not counterbalanced by NTs’ rabies virus receptor—enhancing property and viral uptake. Adult mouse dorsal root ganglion cultures can be regarded as being a useful model for detecting therapeutic targets and evaluating experimental antiviral drugs.

Differential use of the nicotinic receptor by rabies virus based upon substrate origin

To determine the role that the neuronal nicotinic acetylcholine receptor plays in the adsorption process of rabies virus (RV), adult dorsal root ganglion dissociated cultures were exposed to nicotinic agonists before being inoculated. The fixed strain of RV Challenge Virus Standard-11 (CVS-11) was used after being passaged in two different ways, in baby hamster kidney (BHK) cells and in adult mouse brain (MB). Carbachol and nicotine reduced the percentage of CVS-MB infected neurons, yet none of the agonists tested changed the proportion of CVS-BHK infected neurons. This result suggests that the RV phenotype changes depending on its replication environment and neuronal nicotinic acetylcholine receptors are preferentially used for infection by RV strains adapted to adult mouse brain but not to fibroblasts.

An anterograde degeneration study of the distribution of regenerating rat myelinated fibers in the silicone chamber model.

Specificity of reinnervation after a peripheral nerve lesion has given rise to considerable controversy. As a contribution to solving this issue we have evaluated the specificity of reinnervation of the peroneal nerve after a complete transection of the sciatic nerve repaired with an 8 mm silicone tube, leaving a 4 mm gap between the nerve stumps. Our findings reveal unspecificity of reinnervation of the distal peroneal branch. This lack of specificity is shown by a random distribution of fibers originating from both proximal branches at the level of the tube and at distal peroneal and tibial branches, argue against specificity of regeneration in this model.

Is S-100 protein a suitable marker for adult Schwann cells?

Dear Editor: Proteins belonging to the S-100 family are calcium-binding, EFhand type molecules, widely distributed in different tissues. Among the various functions that have been described for these proteins, specifically for S-100~, are the following: modulation of other proteins in a calcium-dependent manner, promotion of neurite extension, mitogenesis, and thus proliferation of glial cells. The regulation of S-100 levels may be very complex. Increased levels of S-100 have been related to abnormal cellular growth in some cellular tumors such as melanoma, myelocyte leukemia, and thyroid carcinoma. On the other hand, it has been reported that astrocytes and skeletal muscle cells increase the expression of S-100 during differentiation (Zimmer et al., 1995). S-100 protein has been widely used as a Schwann cell marker both in vivo and in vitro (Spreca et al., 1989; Bolin et al., 1992; De Deyne et al., 1994; Gu et al., 1995). Many in vitro studies regarding Schwann cells have been developed using different cellular sources. These cells are extracted from sensorial ganglia and peripheral nerves of animals from different species and ages (embryos, newborns, and adults) by tissue dissociation or explants and cultured under different conditions such as differential adhesion, differential growth, mitogens, antimitogenic agents, and complements (Armati et al., 1990; Morgan et al., 1991; Morrissey et al., 1991; Bolin et al., 1992; Rutkowski et al., 1992; Chi et al., 1993; De Deyne et al., 1994; Haynes et al., 1994; Peulve et al., 1994; Zhang et al., 1994; Ansselin et al., 1995). An immunocytochemical technique for the detection of S-100 was used in our study during the standarization of the in vitro culture of dorsal root ganglia (DRG) Schwann cells from adult mice, in order to identify glial ceils. However, regarding S-100 protein, two different subpopuiations are present in these cultures according to the results shown below. Schwann cell cuhures were established from DRG of adult albino mice, strain ICR, ranging from 8 to 10 wk old, and weighing approximately 30 g. The mice were maintained at the animal facility of the Instituto Nacional de Salud in Bogotzl. The methodology applied in this study included procedures previously reported (Manthorpe and Varon, 1989; Morgan et al., 1991; Rutkowski et al., 1992; Carroll et al., 1993; Morrissey et al., 1995). Animals were sacrificed by cervical dislocation. Ganglia were dissected under sterile conditions, and their remaining root nerves were eliminated. The ganglia were then digested with 128 U/ml of collagenase incubated in Dulbecco modified Eagle medium (DMEM) for 1 h at 37 ~ C. Enzymatic digestion of tissue was followed by mechanical dissociation using a narrow-end Pasteur pipette. The resulting cellular suspension was centrifuged, and the cell pellet was resuspended in complete medium (DMEM + 10% FBS + 0.1 ~g/ml cholera toxin + 2 ~M forskolin) and antibiotics (100 U/ml penicillin and 100 ~g/ml streptomycin) and cultured in 25 cm 2 bottles previously treated with 10 mg/ml type I collagen. Prior to incubation, cellular viability was determined by the trypan blue dye exclusion method (Freshney, 1988). The medium was changed every other day until about 15 d later, when the cultures reached confluency. Cells were subcuhured using 560 U/ml trypsine in two 25cm 2 culture bottles as described earlier (first passage). The second and third passages were carried out using the same procedure as well, and cells obtained from each passage were used for immunocytochemical evaluation. Cells obtained from the third passage were detached and seeded (20,000 cells/well) onto 24-well plates containing round coverslips previously treated with collagen. Plates were incubated for 24 h in complete medium, washed with sernm-free medium and with buffer, fixed with 4% paraformaldehyde, and processed for immunocytochemistry. Anti-S-100 antibodies recognizing both alpha and beta isoforms (Dako, ICN, Lipsaw) were used for a first series of experiments (Table 1), and four wells were processed for GFAP immunodetection. In a second series of experiments (follow-up), the procedure was the same as the one described earlier, except for a slight variation of using a parallel 24-well plate in order to evaluate the immunocytochemical activity (using Dako antibody) for each cultured age, as shown in Table 2. The following alternatives for each step of the procedure were tested in order to determine the best choice: (1) buffers (phosphate buffer pH 7.4, PBS pH 7.3, and Tris-Triton pH 8.6), (2) endogenous peroxidase inactivators (0.3% H202 in PBS, in buffer Tris-Triton, and in methanol), (3) primary anti-S100 polyclonal antibodies (Dako) at 1/250 and 1/500 dilutions; Lipsaw, at a predetermined dilution; and ICN, at 1/300 and 1/500 dilutions, according to each manufacturers recommendations, and (4) incubation periods (1 h at 37 ~ C or overnight at 4 ~ C). In general terms, the cultures were washed in buffer and permeabilized with 0.1% Triton X-100 in buffer (except when buffer Tris-Triton containing 0.5% Triton X-100 was used). The endogenous peroxidase was inactivated with 0.3% H202 and the specific sites were blocked with 3% horse serum. Cultures were then incubated with the primary antibody prepared in the blocking solution; ceils were washed and incubated with the biotinylated secondary antibody (1:200 in buffer) for 30 min at 37 ~ C. Again, cells were washed and incubated with the avidin-biotin-peroxidase complex (ABC Vectastain kit) for 45 min. The reaction was developed with a 1:1 0.02% H202 solution in distilled water and 0.01% diaminobenzidine in Tris pH 7.3, or Tris-imidazol pH 7.6. The development of the reaction was followed up using a light microscope. After development, cultures were washed with distilled water and stained with Meyer Hemalun, dehydrated, and mounted in Permount. Wells processed without the addition of primary antibodies