molecules & cells
1. Glatiramer Acetate Treatment Positively Influences In Vitro Astrocyte Proliferation.
Zanon, RG; Oliveira, ALR
Anatomy Department of the Biology Institute, UNICAMP, Campinas
Glatiramer acetate (GA) has been indicated for reduction of the frequency of the relapses in patients with remittent/recurrent multiple sclerosis. Although effective, the GA’s therapeutic mechanism(s) is (are) not totally clear. However, studies based on the experimental autoimmune encephalomyelitis (EAE) have shown that GA displaces MBP peptides from the MHC class II antigen presentation molecules, inhibiting T cell activation involved in the onset of EAE. Besides its immunomodulatory action, GA may probably exert effects on the nervous tissue itself. Such interactions are currently poorly understood. In the present work we have studied the effect of different doses of GA (1.2 , 2.5 e 5.0 µg/ml of medium) during 5 consecutive days on astrocyte primary cell cultures, regarding cell multiplication and reactivity. After treatment, the cultures were imunolabeled with anti-GFAP and counterstained with DAPI for quatification of the number cells and degree of astrogliosis. Our results showed that GA treatment increased cell proliferation, specially at 5.0 µg/ml, similarly to what has been described to T helper 2 and 3 cells. However, the measurement of GFAP immunolabeling did not show any significant difference among the experimental groups, indicating that GA does not induce astrocyte hypertrophy. This fact is consistent with recent in vivo results with EAE model and indicates that part of the positive outcome of GA treatment may rather involve astrocyte proliferation, leading to the upregulation of local neurotrophic synthesis.
2. DYE COUPLING MEDIATED BY RADIAL GLIA IN THE SUBVENTRICULAR ZONE.
1Freitas, A. S. **; 1Furtado, C. M. **; 1Menezes, J.R.L. ; 2Hedin-Pereira C ; 1Fróes, M. M.
1, Anatomia - CCS, UFRJ; 2 Instituto Biofísica Carlos Chagas Filho, UFRJ;
Objective: Radial glial cells (RG) are multipotent precursors present during SNC embryonic development and are characterized by a bipolar radial morphology, with processes that expand the whole width of the neural tube wall. After birth these cells populate the subventricular zone (SVZ), an important postnatal neurogenic region. We have shown previously that the early postnatal SVZ cells display extensive junctional coupling. In this study, we have analyzed the specific contribution of the RG for this coupling. To specifically target RG as sources of dye-coupling in situ, we have developed a new technique that involves direct cell loading through processes that reach the pial surface, using a mix of gap junction permeant and non permeant fluorochromes. Methodology and Results: Rat brains with 1, 4 and 6 post-natal days (P1, P4, P6) were immersed in PBS ØCa+2/EGTA/glucose. Their pial surfaces were scratched and a mix of fluorochromes, consisting of the gap junction permeant Lucifer Yellow (LY, 443Da) and the non permeant, Rhodamine-conjugated Dextran (RD, 3KDa), that were intracellularly loaded through the ruptured cell processes. After wash (Gey’s salt solution) the brains were fixed (PFA 4%) for 3h. Some were cryoprotected for cryostat sectioning. The sagittal slices were analyzed under epifluorescence for identification of directly loaded cells (LY+RD+) and coupled ones (LY+RD-) in the SVZ. Cells LY+RD+ in the SVZ were found restricted to the underlying pial loading surface. The retrogradely labeling of these cells with carbocianines placed in the pial surface confirms their RG identity. Coupled cell profiles (LY+RD-) were distributed in a bimodal pattern although more frequently present localized to the SVZ external portion. Although the LY+RD- cells were not positive for the neuroblast marker, beta-tubulin III, the morphological diversity of coupled cells were suggestive of heterocellular coupling partnerships. In addition, coupled cell profiles in the SVZ were restricted to the cortical domain, with an abrupt stop in the striatum border, probably indicating the presence of a functional border between these two germinative regions. Conclusions: The methodology herein developed was suitable to specifically dissect RG cells as SVZ gap-junction permeant sources for dye coupling studies in this neurogenic niche. We have demonstrated the direct participation of RG in extensive cell coupling within the SVZ. Moreover, the spatial distribution of coupled cell profiles strongly highlights the importance of studying these coupled syncytia in the context of neurogenesis regulation and regional specialization that possibly drives the postnatal development of the SVZ. Financial Support: CAPES, CNPq, FAPERJ, FUJB, PADCT, PRONEX
3. Neuronal and ependimocyte types in the cerebral cortex of the tropical lizard Tropidurus hispidus: A Golgi study.
Jean-Marcell de Azevedo Mota Nunes1; Fabrício Tavares Cunha de Almeida1; José Ronaldo dos Santos1; Márcia Leite dos Santos1; Carlos Lopez-Garcia2; Assuncion Molowny2; Murilo Marchioro1.
1Laboratório de Neurofisiologia, Departamento de Fisiologia, Universiade Federal de Sergipe, São Cristóvão-SE, Brasil; 2Laboratório de Neurobiologia Celular; Universitat de València, Espanha.
In this work we described the neuronal and ependymocyte types in the cerebral cortex of the lizard, T. hispidus. Animals were captured in the surroundings of the University campus and were maintained in a terrarium. All experiments were approved by the local Ethical Comitee. Under deep thiopental anesthesia, the animals were transcardially perfused with 25% gluataraldehyde and 4% paraformaldehyde in phosphate buffer (0.2 M; pH=7.4). The brains were removed and kept overnight in the same fixative at 4oC. For staining, the brains were left 2-7 days in a mixture of 2.4% potassium dichromate and 5% glutaraldehyde, followed by 1-4 days of impregnation in 0.75% silver nitrate. After impregnation, 125-200 µm transverse sections were obtained and embedded in Araldite between two cover glasses. Selected cells were photographed and classified. From a total of 735 impregnated cells, thirteen types of neurons were identified and classified into four categories according to dendritic pattern and shape of the soma. The granular multipolar was the most frequent neuron (48%), followed by the granular bipyramidal type (26%). Most impregnated neurons were found in the dorsal (40%) and dorso-medial cortices (37.5%). A variety of ependymocyte types were revealed in the T. hispidus cortex. They were found in the ependyma, inner and outer plexiform layers. They showed one, two, three or many cilia projecting from the soma to the ventricle and have smooth, sparsely spiny or heavily spiny process projecting to the inner plexiform layer (short), cellular layer (medium) or outer plexiform layer (large). The knowledge of neuronal and ependymocyte types in the T. hispidus cortex will support further electrophysiological studies related to postnatal neurogenesis and neuronal regeneration.
4. glycogen stores IN hypothalamus of malnourished rats. Tenório1, F.; dos Santos1, M.C., Lima1, S. S.; Sinder1 MP; Moura2, A. S.; Barradas1, P. C. 1Farmacologia e Psicobiologia, UERJ; 2Ciências Fisiológicas, UERJ.
Glucose is brain´s most important energy source. Glycogen stores are present in neurons and glial cells during development and could be recruited during intense and acute demands. Animals submitted to malnutrition during a restricted period of their development presented alterations on the expression/distribution of hypothalamic molecules related to energy metabolism. We previously observed a delay in nitric oxide expression in hypothalamus. In this work we analyzed the effects of a hipoproteic diet applied to lactating rats during the first ten days of life in hypothalamic glycogen stores.
Animals from P10 to P45, normal and malnourished, were anaesthetized and fixed by perfusion with 4% paraformaldehyde. The methodology modified from Rosenberg and Dichter to detect glycogen were performed in brain slices of 30mm. To identify the stained profiles, immunohistochemistry were realized with a polyclonal anti-GLUT2 antibody revealed with a secondary antibody conjugated to Cy3. In malnourished animals we observed a great reduction on glycogen stores mainly from P10 to P30. By P45, staining for glycogen is very faint and no difference could be observed. The staining presented a radial profile on third ventricle border mainly in the arcuate nucleus and median eminence. These radial profiles were GLUT2 positive indicating that these cells were tanycytes. Our work suggests that glycogen stores are being depleted in malnourished rats in response to a decrease in macronutrients. The lack of glycogen during development of the hypothalamic circuitry may be causing a delay in the expression of molecules involved in the regulation of energy metabolism.
5. ABL-DEPENDENT APOPTOSIS IN RB-/- EMBRYOS.
Helena L. Borges1,2, Irina Hunton1, Jean Y.J Wang1.
1University of California San Diego, La Jolla, CA 92093-0820, US. 2Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, 21.941-590, Brazil.
The retinoblastoma protein RB regulates cell proliferation, differentiation and apoptosis. Homozygous knockout of Rb in mice causes embryonic lethality associated with ectopic proliferation and excessive apoptosis especially in the developing nervous system. RB binds to a number of cellular proteins including the Abl protein and inhibits its tyrosine kinase activity. Ex vivo experiments have shown that genotoxic or inflammatory stress can activate Abl kinase to stimulate apoptosis. To test if Abl contributes to the defects seen in Rb-null mice embryos, we combined the Abl-null allele with the Rb-null allele. We conducted a large scale mating of double heterozygous Abl+/- Rb+/- mice; 1029 embryos were collected and examined between E10 and E14.5. We have shown that the genetic ablation of Abl can reduce apoptosis in the developing central nervous system, but not in the peripheral nervous system. Heterozygosity of the Abl loci also delayed the death of Rb-null embryos, although it did not result in live birth. These results are consistent with the inhibitory interaction between RB and Abl. Moreover, the results provide in vivo evidence for the pro-apoptotic function of Abl, highlighting differences between central and peripheral nervous system.
6. Antioxidant effect of levetiracetam in mice brain.
Oliveira AAb, Linhares, MIb, Carvalho, AMRb, Moura BAb, Aguiar, LMVb, Viana, GSBb, Sousa, FCFb, Fonteles MMFa,b.
aDepartment of Pharmacy, bDepartment of Physiology and Pharmacology, Laboratory of Neuropharmacology, Federal University of Ceará, Fortaleza, Brazil.
Levetiracetam (LEV) is a new antiepileptic drug effective as adjunctive therapy for partial seizures and in experimental models of seizures, including pilocarpine-induced seizures. Objectifying to investigate if LEV has antioxidant properties, in vitro antioxidant assays were made measuring the production of thiobarbituric acid reactive substances (TBARS), as indicator of lipid peroxidation, and nitrite-nitrate level. Swiss mice (25-30g) were decapitated and its brains were removed rapidly at 4 C and stored for 24h at 20 C (Auddy et al., 2003). The whole brain besides the cerebellum was homogenized in 50mM potassium phosphate buffer (pH 7.4) and the concentration adjusted to 1g wet weight of brain per 60 mL. After that 250 L of the homogenate in the absence (control) or presence of LEV (50, 100 and 200 g/mL) was obtained for neurochemistry assay. Measurement of TBARS was determined according Agar et al (1999) and nitrite-nitrate determination according to Green et al (1981). The results showed an increase in the lipid peroxidation and nitrite-nitrate contents in the brains submitted to oxidative stress (stress group) when compared control group (TBARS- Control: 0.87+0.09; Stress group: 2.70+0.43, n=10; Nitrite- Control: 0.84+0.08; Stress group: 57.73+5.90, n=7). Otherwise, LEV significantly diminished, in all tested concentrations, both TBARS and nitrite contents when compared to stress groups (p<0.01) (TBARS- LEV200: 1.42+0.24; LEV100: 1.42+0.28; LEV50: 0.98+0.20, n = 11-13; Nitrite- LEV200: 45.52+1.94; LEV100: 27.76+1.22; LEV50: 34.61+1.70, n=7). These results show an antioxidant activity of LEV which might contribute to the drug ability to behave as a possible neuroprotective agent. Supported by CNPq.
7. STRESS-INDUCIBLE PROTEIN 1 (STI-1) - INTRACELLULAR TRAFFICKING AND INTERACTION WITH CELLULAR PRION PROTEIN (PrPc)
Caetano, F A1; Magalhaes, A C1; Hajj, G N2; Lopes, M H2; Massensini, A R3; Prado, V F4; Martins, V R2; Prado, M A M1. 1Depto. de Farmacologia, 3Depto. de Fisiologia e Biofísica, 4Depto. de Bioquímica e Imunologia - UFMG, 2Instituto Ludwig de Pesquisa para o Câncer -São Paulo.
The STI-1 is a co-chaperonine and it also may function as a secreted neurotrophic factor. Previous work has shown that recombinant PrPc interacts with STI-1 promoting neuritogenesis and neuroprotection by distinct signaling pathways. In order to understand possible physiological functions of STI-1 and PrPc we investigate their trafficking. A functional fluorescent STI-1 and a STI-1 without PrPc binding site (STI-1) were generated to follow protein trafficking by fluorescence microscopy. Fluorescent STI-1 binds to cells in a saturable and specific way and after 10-20 minutes of cell contact the protein is internalized and accumulates in endocytic organelles. Co-localization studies with markers of sub-cellular organelles indicated that fluorescent STI-1 is internalized to flotillin-1 positive vesicles and is then found in Rab7 positive acidic organelles. In contrast, by using markers of endossomes and coated-pits, we found no evidence for the participation of the classical clathrin-mediated endocytic pathway in the internalization of STI-1. SN56 cells transfected with PrPc-GFP were perfused with fluorescent STI-1 or STI-1. The results have shown that STI-1, no STI-1, promotes the internalization of PrPc-GFP that is endocyting by distint pathway from STI-1. These results suggest that STI-1 is endocyted by a clathrin independent pathway and your interaction with PrPc in cellular membrane lead to internalization of the cellular prion protein. Supported by: CNPq e FAPESP
8. The neuronal factor rhPIF/DCD protects rats against the neurotoxic effect of kainic acid by antiapoptotic pathway.
Persike, D. S.; Cipolla-Neto, J.; Belizário, J.E.
University of Sao Paulo.
PIF/DCD (proteolisys-inducing factor/dermcidin) is a protein expressed in several human tissues including the brain. It is overexpressed in pathological conditions as in breast carcinomas and, the in vitro studies revealed that PIF/DCD expression has been associated with growth and cellular survival. Peptides arising from PIF/DCD proteolytic processing are produced by mononuclear cells of peripheral blood during the early life. It has been found in the cortical and hippocampal neurons, but its physiological function remains unknown. Previous studies employing this protein in in vivo and in vitro experiments showed that PIF/DCD act as neural survival factor. Here, we evaluated the protective effect of the recombinant human protein rhPIF/DCD against lesion caused as consequence of status epilepticus (SE) induced by kainic acid in rats (15 mg/kg, i.p.). Our results show that pretreatment with rhPIF/DCD (30-260 g/kg, i.v.) decreases significantly the severity of the SE. Besides, a significant reduction in the DNA fragmentation and in the caspases 2, 3 and 6 activities were also observed in different brain regions of rats studied 3 hours after SE initiation. These results showed that the pretreatment with rhPIF/DCD, is able to protect the central nervous system against the neurotoxic effect of the kainic acid, and this effect can involve the caspases cascade inhibition.