Indirect orthodontic bonding - a modified technique for improved efficiency and precision

INTRODUCTION: The indirect bonding technique optimizes fixed appliance installation at the orthodontic office, ensuring precise bracket positioning, among other advantages. In this laboratory clinical phase, material and methods employed in creating the transfer tray are decisive to accuracy. OBJECTIVE: This article describes a simple, efficient and reproducible indirect bonding technique that allows the procedure to be carried out successfully. Variables influencing the orthodontic bonding are analyzed and discussed in order to aid professionals wishing to adopt the indirect bonding technique routinely in their clinical practice. .

INTRODUÇÃO: a técnica de colagem indireta prioriza a otimização do procedimento de montagem do aparelho fixo na clínica ortodôntica, assegurando, entre outras, vantagens relacionadas à precisão no posicionamento dos braquetes. Nesse procedimento clínico laboratorial, o material e o método de confecção da moldeira de transferência são determinantes no quesito precisão. OBJETIVO: este artigo descreve uma técnica de colagem indireta simples, eficiente e reprodutível, para que o procedimento possa ser realizado com sucesso. Variáveis que exercem influência sobre o procedimento são analisadas e discutidas, a fim de auxiliar o profissional a adotar, de forma rotineira, a técnica de colagem indireta em sua prática clínica. .

Isolamento e cultivo de neurônios e neuroesferas de córtex cerebral aviar / Isolation and culture of neurons and neurospheres from chicken brain cortex

Métodos de cultivo celular são convenientes na realização de análises funcionais de alterações/interações protéicas das células neuronais, auxiliando a decifrar o interactoma de proteínas chaves na neurogênese de doenças do Sistema Nervoso Central. Por esse motivo, culturas de neurônios e neuroesferas isolados do córtex cerebral aviar representam um modelo acessível para o estudo de diversas doenças neurológicas, tal como a epilepsia. A espécie aviar apresenta peculiaridades em seu proteoma neuronal, visto a presença de uma expressão diferenciada de proteínas chaves no metabolismo energético cerebral, algumas destas (VDAC1 e VDAC2) desempenham papel importante na compreensão do mecanismo da epilepsia refratária. A metodologia estabelecida no presente estudo obteve cultivo de neuroeferas, onde as células cresceram tipicamente em aglomerados atingindo, dentro de 7 dias, o diâmetro ideal de 100-200 µm. A diferenciação celular das neuroesferas foi obtida após a aderência destas às placas tratadas com poli-D-lisina, evidenciada pela migração de fibras do interior da neuroesfera. Ao contrário das neuroesferas, os neurônios em cultivo extenderam seus neuritos após 11 dias de isolamento. Tal modelo in vitro pode ser utilizado com sucesso na identificação das variáveis neuroproteômicas, propiciando uma avaliação global das alterações dinâmicas e suas interações protéicas. Tal modelo pode ter aplicações em estudos dos efeitos de indutores da morte celular e bloqueadores de canais de membrana mitocondriais em proteínas chaves do metabolismo energético cerebral.

Cell culture methods are used for studies of protein interactions in neural cells, helping to detect the interactome of proteins linked to generation of central nervous system diseases. For this reason, neural cells and neurospheres isolated from cortical chicken brain are a current model for studies of neurological diseases, such as epilepsy. Chicken brain has key characteristics on its proteome, with a differential expression of proteins linked to energy metabolism, some of them (VDAC 1 and VDAC 2) play an important role in understanding mechanism of refractory epilepsy. Using the methods described, we found neurospheres, in which cells grow in structures with the ideal diameter of 100-200µm within seven days after isolation. Neurospheres differentiation was obtained after adhesion of these cells to surfaces coated with poly-D-Lysine, detected by migration of fibers inside them. Unlike neurospheres, neurons extended neurites after 11 days of isolation. Here we describe a method to isolate and culture neurons and neurospheres from chicken cerebral cortex. Such "in vitro" model can be utilized on studies of neuronal protein differential expression and interaction. Cultures of isolated neurons represent an accessible model on studies of apoptosis and channel blockers of key proteins linked to brain metabolism.

Nestin down-regulation of cortical radial glia is delayed in rats submitted to recurrent status epilepticus during early postnatal life / Atraso no desaparecimento da nestina na glia radial cortical de ratos submetidos a recorrentes status epilepticus durante o desenvolvimento pós-natal precoce

OBJECTIVE: Nestin is temporarily expressed in several tissues during development and it is replaced by other protein types during cell differentiation process. This unique property allows distinguishing between undifferentiated and differentiated cells. This study was delineated to analyze the temporal pattern of nestin expression in cortical radial glial cells of rats during normal development and of rats submitted to recurrent status epilepticus (SE) in early postnatal life (P). METHOD: Experimental rats were submitted to pilocarpine-induced SE on P7-9. The cortical temporal profile of nestin was studied by immunohistochemistry at multiple time points (P9, P10, P12, P16, P30 and P90). RESULTS: We observed delayed nestin down-regulation in experimental rats of P9, P10, P12 and P16 groups. In addition, few radial glial cells were still present only in P21 experimental rats. CONCLUSION: Our results suggested that SE during early postnatal life alters normal maturation during a critical period of brain development.

OBJETIVO: A nestina, temporariamente expressa em diversos tecidos durante o desenvolvimento, é substituída no processo de diferenciação celular, o que permite a distinção entre células diferenciadas e indiferenciadas. O objetivo deste estudo foi verificar o padrão temporal da expressão da nestina nas células da glia radial cortical de ratos durante o desenvolvimento normal e nos ratos submetidos a sucessivos status epilepticus (SE) no periodo pós-natal precoce (P). MÉTODO: Os animais foram submetidos ao SE induzido pela pilocarpina em P7-9. O perfil temporal da nestina foi estudado por imuno-histoquímica em P9, P10, P12, P16, P30 e P90. RESULTADOS: Nos ratos experimentais, observamos atraso no desaparecimento da nestina nos grupos P9, P10, P12 e P16. Ainda, encontramos algumas glias radiais corticais apenas em P21 experimental. CONCLUSÃO: Nossos resultados sugerem que o SE durante o desenvolvimento pós-natal precoce altera o processo de maturação durante um periodo crítico do desenvolvimento encefálico.

Asymmetrical release of interleukin-6 by cultured cerebral cortical astrocytes treated with lipopolysaccharide.

BACKGROUND & OBJECTIVES: The distribution of brain interleukin-6 (IL-6) may be asymmetrical both in cortex and hippocampus. While the brain asymmetry has been extensively investigated, the cellular origin of asymmetrical cytokine induction in the cortex has not been addressed. It was hypothesized that the immune function of glia cell to the inflammatory insults is asymmetrically distributed in the two brain hemispheres. To test this hypothesis, we examined the IL-6 secreting ability of the astrocytes in both the left and right neocortex treated with lipopolysaccharide(LPS) cultured in vitro. METHODS: Two groups of astrocytes cultured in vitro from the two cerebral cortices of the neonatal BALB/ c mice were selected and experimental group was treated with LPS (10 mug/ml) for 24 h. IL-6 levels were measured in both LPS-treated and untreated astrocytes. To confirm the gene array data on the secretion of IL-6 by cortical astrocytes in the left and right hemispheres, semi-quantitative reverse transcriptionpolymerase chain reaction (RT-PCR) was conducted. RESULTS: A statistically significant difference between the levels of IL-6 in cortical astrocytes in the left and right hemispheres of culture supernatants was observed (P<0.05). Cortical astrocytes in the left hemisphere had significantly increased IL-6 mRNA levels compared with cortical astrocytes in right hemisphere (P<0.05). INTERPRETATION & CONCLUSION: The results showed asymmetrical release of brain IL-6 by cerebral cortical astrocytes to the inflammatory insults both in protein and mRNA levels.

In vitro study on bFGF expression of rat astrocytes after mechanical injury / 法医学杂志

OBJECTIVE@#To study the alteration of basic fibroblast growth factor (bFGB) expression in astrocytes in vitro after mechanical injury and to understand the repair mechanism of brain injury.@*METHODS@#Astrocytes were isolated from cerebral cortex of SD rats born in 24 hours, and then cultured and purified. The cultured astrocytes were randomly divided into control group and injury groups that were subjected to mechanical injury at 30 min, 1h, 3h, 6h, 12h, 24h, 3d, and 7d. The levels of bFGF expression in the astrocytes after injury were detected by ABC immunohistochemistry.@*RESULTS@#More than 95% of the cultured cells were astrocytes. The levels of bFGF expression werevery low in the control group. On the other hand, increased levels of bFGF expression could be observed at 1-3h after injury. The expression levels increased significantly at 6-12h, reached peak level at 24h, remained at the high level up to 3 days, and the decreased gradually.@*CONCLUSION@#The changes of bFGF expression levels in cultured astrocytes in vitro after mechanical injury are similar to that observed in vivo experimental model, both of which show time-dependant characteristic, with only slightly earlier expression of bFGF observed in vitro. Thus, the expression of bFGF after injury can be one of evidences for estimation of brain injury intervals. the cell injury model in vitro may have superiority in the study of the molecule mechanism of tissue and cell injury.

Bone morphogenetic protein 4 stimulates neuronal differentiation of neuronal stem cells through the ERK pathway

Bone morphogenic protein 4 (BMP4), a member of the TGF-beta superfamily, induced neural differentiation of neural stem cells (NSCs) grown in a medium containing basic fibroblast growth factor (bFGF). The Ras protein level and the activities of the downstream ERKs were increased by transfection of BMP4 or treatment with recombinant BMP4. The effects of BMP4, including activation of the Ras-ERK pathway and induction of the neuron marker beta-tubulin type III (Tuj1), were blocked by co-treatment of the BMP4 antagonist, noggin. The roles of the Ras-ERK pathway in neuronal differentiation by BMP4 were revealed by measuring the effect of the ERK pathway inhibition by dominant negative Ras or PD98059, the MEK specific inhibitor. BMP4 is a transcriptional target of Wnt/beta-catenin signaling, and both the mRNA and protein levels of BMP4 were increased by treatment of valproic acid (VPA), a chemical inhibitor of glycogen synthase kinase 3beta (GSK3beta) activating the Wnt/beta-catenin pathway. The BMP4- mimicking effects of VPA, activation of the Ras-ERK pathway and induction of Tuj1, also were blocked by noggin. These results indicate the potential therapeutic usage of VPA as a replacement for BMP4.

Small-molecule aggregation inhibitors reduce excess amyloid in a trisomy 16 mouse cortical cell line

We have previously characterized a number of small molecule organic compounds that prevent the aggregation of the β-amyloid peptide and its neurotoxicity in hippocampal neuronal cultures. We have now evaluated the effects of such compounds on amyloid precursor protein (APP) accumulation in the CTb immortalized cell line derived from the cerebral cortex of a trisomy 16 mouse, an animal model of Down's syndrome. Compared to a non-trisomic cortical cell line (CNh), CTb cells overexpress APP and exhibit slightly elevated resting intracellular Ca2+ levéis ([Ca2+]¡). Here, we show that the compounds 2,4-dinitrophenol, 3-nitrophenol and 4-anisidine decreased intracellular accumulation of APP in CTb cells. Those compounds were non-toxic to the cells, and slightly increased the basal [Ca2+]¡. Results indícate that the compounds tested can be leads for the development of drugs to decrease intracellular vesicular accumulation of APP in trisomic cells.

Protective Effect of Ultra Low Molecular Weight Heparin on Glutamate-Induced Apoptosis in Cortical Cells

PURPOSE: To investigate the effect of ultra low molecular weight heparin (ULMWH) on glutamate induced apoptosis in rat cortical cells and to explore the possible mechanisms. MATERIALS AND METHODS: Cell viability was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Apoptosis was first analyzed with Hoechst 33258 and then confirmed by DNA fragmentation. The concentration of free intracellular calcium ([Ca2+](i)) was determined with fura-2/AM fluorometry. The expression of Bcl-2 family protein and caspase-3 were evaluated with Western blot. RESULTS: Typical apoptotic morphological change in rat cortical cells treated with 100micromol/L glutamate for 24h was detected by Hoechst 33258 staining, which was then confirmed by the DNA ladder of agarose gel electrophoresis. The apoptotic rate of the glutamate treated cells was up to 33.21%, and 24 h of treatment with glutamate increased [Ca2+](i), down-regulated Bcl-2 expression, up-regulated Bax expression, and increased caspase-3 activation in rat cortical cells. Our research demonstrated that ULMWH pretreatment can prevent the glutamate- induced apoptosis, attenuate the increase of [Ca2+](i) not only in medium containing Ca2+ but also in Ca2+-free medium, up-regulate the expression of Bcl-2, down-regulate the expression of Bax, and decrease caspase-3 activation. CONCLUSION: ULMWH has neuroprotective capacity to antagonize glutamate-induced apoptosis in cortical cells, through decrease of Ca2+ release and modulation of apoptotic processes.

Antioxidant responses of cortex neurons to iron loading

Brain cells have a highly active oxidative metabolism, yet they contain only low to moderate superoxide dismutase and catalase activities. Thus, their antioxidant defenses rely mainly on cellular reduced glutathione levels. In this work, in cortical neurons we characterized viability and changes in reduced and oxidized glutathione levels in response to a protocol of iron accumulation. We found that massive death occurred after 2 days in culture with 10 mM Fe. Surviving cells developed an adaptative response that included increased synthesis of GSH and the maintenance of a glutathione-based reduction potential. These results highlight the fundamental role of glutathione homeostasis in the antioxidant response and provide novel insights into the adaptative mechanisms of neurons subjected to progressive iron loads.

Isolation, culture and identification of neural stem cells in new-born rats / 华中科技大学学报（医学）（英德文版）

The cortexes were obtained from new-born rats and dissociated to single cells by triturating. The cells were cultured in neural stem cell (NSC) culture medium (DMEM supplemented with bFGF, EGF and B27) and formed primary neurospheres after 7 days. Single cells dissociated from neurosphere were cultured in 96-well plates and formed single-cell cloning neurosphere 7 days later. The primary and single-cell cloning neurospheres were both positive for the immunofluorescent staining of nestin and were identified as NSC. It was proved that NSC can be expanded in vitro and provide seed cells for neural tissue engineering.

The 9-O-acetyl GD3 gangliosides are expressed by migrating chains of subventricular zone neurons in vitro

Neurons from the anterior subventricular zone (SVZ) of the cerebral cortex migrate tangentially to become interneurons in the olfactory bulb during development and in adult rodents. This migration was defined as neuronophilic, independent of a radial glial substrate. The cortical SVZ and the rostral migratory stream to the olfactory bulb were shown to be rich in 9-O-acetyl GD3 gangliosides (9-O-acGD3), which have been previously shown to be implicated in gliophilic migration in the rodent cerebral cortex and cerebellum. In the present study, we performed SVZ explant cultures using rats during their first postnatal week to analyze the expression of these gangliosides in chain migration of neuronal precursors. We characterized migrating chains of these neuroblasts through morphological analysis and immunocytochemistry for the neural cell adhesion molecule. By using the Jones monoclonal antibody which binds specifically to 9-O-acGD3 we showed that migrating chains from the SVZ explants express 9-O-acGD3 which is distributed in a punctate manner in individual cells. 9-O-acGD3 is also present in migrating chains that form in the absence of radial glia, typical of the neuronophilic chain migration of the SVZ. Our data indicate that 9-O-acetylated gangliosides may participate in neuronophilic as well as gliophilic migration

Expression of CXCR4 in the brain of feline immunodeficiency virus infected cat.

Advances in understanding the mechanisms of human immunodeficiency virus (HIV)-1 entry have revealed that the cell surface CD4 expression alone is insufficient and needs an additional molecule on its surface for the viral entry. These are G-protein coupled seven transmembrane (7-TM) family molecules (chemokine receptor) and amongst them one is CXCR4. Feline homologue of CXCR4 acting as a co-receptor for feline immunodeficiency virus (FIV) entry is already reported for the Crandle feline kidney cells strain (CrFK) of FIV. An experiment was carried out to search the expression of CXCR4 retrospectively in FIV (CrFK) infected cat brain tissues using immunohistochemically in the formalin fixed paraffin sections against 12G5, a mouse monoclonal antibody to CXCR4. We observed the expression of this receptor in feline neurons, astrocytes and in some vascular endothelial cells. The study of expression of CXCR4 in the brain, which is one of the many chemokine receptors in the central nervous system, may provide further insight into the interactions between brain cells, pathogens, and the immune system, and help understand the pathogenesis of HIV dementia.

SH oxidation stimulates calcium release channels (ryanodine receptors) from excitable cells

The effects of redox reagents on the activity of the intracellular calcium release channels (ryanodine receptors) of skeletal and cardiac muscle, or brain cortex neurons, was examined. In lipid bilayer experiments, oxidizing agents (2,2'-dithiodipyridine or thimerosal) modified the calcium dependence of all single channels studied. After controlled oxidation channels became active at sub microM calcium concentrations and were not inhibited by increasing the calcium concentration to 0.5 mM. Subsequent reduction reversed these effects. Channels purified from amphibian skeletal muscle exhibited the same behavior, indicating that the SH groups responsible for modifying the calcium dependence belong to the channel protein. Parallel experiments that measured calcium release through these channels in sarcoplasmic reticulum vesicles showed that following oxidation, the channels were no longer inhibited by sub mM concentrations of Mg2+. It is proposed that channel redox state controls the high affinity sites responsible for calcium activation as well as the low affinity sites involved in Mg2+ inhibition of channel activity. The possible physiological and pathological implications of these results are discussed.

Glucocorticoid regulation of in vitro astrocyte phagocytosis

Astrocytes participate in central nervous system injury, degenerative diseases and also perform macrophagic functions. The present work investigates: 1) the effect of the physiological glucocorticoid corticosterone (CORT) and the synthetic agonist dexamethasone (DEX) on latex beads phagocytosis by neonatal rat cortical astrocytes in culture, and 2) the expression of immunoreactive glucocorticoid receptors (GR) in astrocytes cultured in different media with or without a pulse application of CORT. The results indicated that glucocorticoids reduced astrocyte phagocytic activity, as occurred with macrophages, independently of the culturing conditions employed. The extent of phagocytosis was inversely related to nuclear immunostaining for GR in cultures in fetal calf serum, which contained endogenous glucocorticoid. However, no correlation was found between nuclear GR and phagocytosis for cultures in glucocorticoid-free medium or in medium containing CORT. It is suggested that additional factors, besides the GR, may be involved in glucocorticoid modulation of astrocyte phagocytosis.

Electrochemical fixation techniques: I. Electrochemical fixation of human brain

An electrochemical brain fixation procedure (EBFP) to treat brains excised from human cadavers is described thoroughly. It is as precise as any other similar method currently available. However, it takes only as much as 36 h to completion instead of the much longer lapses required by immersion in formaldehyde. Actions were taken to secure that it is not a source of artifacts of any kind, neither neurons nor glia or blood vessels. It is, therefore, amenable to be used as a valuable research and teaching tool. Other advantages are that it does not pose any health hazard, is money- and time-saving, and cuts down on equipment and facilities

Patología degenerativa del sistema nervioso. Seminario presentado en el XIX Congreso Latinoamericano de Patología: Enfermedad de Alzheimer / Degenerative conditions of the nervous system: a slide seminar disease of Alsheimer

Mujer de 60 años, quien se volvió inquieta, con delirios de asaltos a su casa, que en el espacio de 6 meses fueron reemplazados progresivamente por apatía. Se notó pérdida progresiva de la memoria y desorientación, que progresó hacia la demencia profunda y caquexia en el espacio de 30 meses. La TAC mostró una atrofia moderada y simétrica que afectaba a todo el cerebro pero más intensamente a los lóbulos frontales. Se realizó biopsia cerebral

Immunocytochemical distribution of gamma isoform of protein kinase C (PKC-gamma) following incomplete ischaemia.

Immunocytochemical distribution of PKC-gamma was examined in rat brain in relation to molecular mechanisms of post-ischaemic neuronal modulation following incomplete ischaemia. Incomplete ischaemia was developed by either permanent occlusion of one common carotid artery (CA) or permanent occlusion of one CA with temporary occlusion of opposite CA. Unilateral CA (UCA) occlusion resulted in a pronounced increase in the intensity of staining and number of PKC-gamma positive neurons in the neocortex ipsilateral to the insult after 3 h. The effect was maximum at 6-12 h and was undetectable after 7 days. CA1 neurons showed an increase immunoreactivity (IR) after 1 day, reached to a peak by 3 days, then reduced to basal levels after 7 days. Bilateral CA (BCA) occlusion showed almost similar changes in the neocortex, but on both sides and short durated. The altered patterns of PKC-gamma IR in the neocortex and hippocampus following CA occlusion may reflect activation and/or down-regulation of PKC-gamma in ischaemic neurons. PKC-gamma may, therefore, potentially play an important role in the post-ischaemic modulation of synaptic efficacy in these neurons and in the neuronal damage following incomplete ischaemia.

Effect of pre and postnatal retinal deprivation on the striate-peristriate cortical connections in the rat

The tangential distribution of the striate-peristriate cortical connections in normal, postnatally eye nucleated and congenitally anophthalmic rats, was studied after a single injection of wheat germ agglutinin conjugated with horseradish peroxidase into the striate cortex. The typical normal pattern of separate fields in the peristriate cortex is altered in eye enucleated animals, in such a way that their areal distribution in the cerebral cortex is increased and each field tends to fuse with the adjacent one. This process is more marked in anophthalmic animals, a finding that is in agreement with the notion that ganglion cells exert their influence before the visual pathway is functional