Glial cell activity is maintained during prolonged inflammatory challenge in rats

We evaluated the expression of glial fibrillary acidic protein (GFAP), glutamine synthetase (GS), ionized calcium binding adaptor protein-1 (Iba-1), and ferritin in rats after single or repeated lipopolysaccharide (LPS) treatment, which is known to induce endotoxin tolerance and glial activation. Male Wistar rats (200-250 g) received ip injections of LPS (100 µg/kg) or saline for 6 days: 6 saline (N = 5), 5 saline + 1 LPS (N = 6) and 6 LPS (N = 6). After the sixth injection, the rats were perfused and the brains were collected for immunohistochemistry. After a single LPS dose, the number of GFAP-positive cells increased in the hypothalamic arcuate nucleus (ARC; 1 LPS: 35.6 ± 1.4 vs control: 23.1 ± 2.5) and hippocampus (1 LPS: 165.0 ± 3.0 vs control: 137.5 ± 2.5), and interestingly, 6 LPS injections further increased GFAP expression in these regions (ARC = 52.5 ± 4.3; hippocampus = 182.2 ± 4.1). We found a higher GS expression only in the hippocampus of the 6 LPS injections group (56.6 ± 0.8 vs 46.7 ± 1.9). Ferritin-positive cells increased similarly in the hippocampus of rats treated with a single (49.2 ± 1.7 vs 28.1 ± 1.9) or repeated (47.6 ± 1.1 vs 28.1 ± 1.9) LPS dose. Single LPS enhanced Iba-1 in the paraventricular nucleus (PVN: 92.8 ± 4.1 vs 65.2 ± 2.2) and hippocampus (99.4 ± 4.4 vs 73.8 ± 2.1), but had no effect in the retrochiasmatic nucleus (RCA) and ARC. Interestingly, 6 LPS increased the Iba-1 expression in these hypothalamic and hippocampal regions (RCA: 57.8 ± 4.6 vs 36.6 ± 2.2; ARC: 62.4 ± 6.0 vs 37.0 ± 2.2; PVN: 100.7 ± 4.4 vs 65.2 ± 2.2; hippocampus: 123.0 ± 3.8 vs 73.8 ± 2.1). The results suggest that repeated LPS treatment stimulates the expression of glial activation markers, protecting neuronal activity during prolonged inflammatory challenges.

Intra-arterial injection of neural stem cells using a microneedle technique does not cause microembolic strokes

Intra-arterial (IA) injection represents an experimental avenue for minimally invasive delivery of stem cells to the injured brain. It has however been reported that IA injection of stem cells carries the risk of reduction in cerebral blood flow (CBF) and microstrokes. Here we evaluate the safety of IA neural progenitor cell (NPC) delivery to the brain. Cerebral blood flow of rats was monitored during IA injection of single cell suspensions of NPCs after stroke. Animals received 1 × 10(6) NPCs either injected via a microneedle (microneedle group) into the patent common carotid artery (CCA) or via a catheter into the proximally ligated CCA (catheter group). Controls included saline-only injections and cell injections into non-stroked sham animals. Cerebral blood flow in the microneedle group remained at baseline, whereas in the catheter group a persistent (15 minutes) decrease to 78% of baseline occurred (P<0.001). In non-stroked controls, NPCs injected via the catheter method resulted in higher levels of Iba-1-positive inflammatory cells (P=0.003), higher numbers of degenerating neurons as seen in Fluoro-Jade C staining (P<0.0001) and ischemic changes on diffusion weighted imaging. With an appropriate technique, reduction in CBF and microstrokes do not occur with IA transplantation of NPCs.

Intracerebroventricularly delivered VEGF promotes contralesional corticorubral plasticity after focal cerebral ischemia via mechanisms involving anti-inflammatory actions

Vascular endothelial growth factor (VEGF) has potent angiogenic and neuroprotective effects in the ischemic brain. Its effect on axonal plasticity and neurological recovery in the post-acute stroke phase was unknown. Using behavioral tests combined with anterograde tract tracing studies and with immunohistochemical and molecular biological experiments, we examined effects of a delayed i.c.v. delivery of recombinant human VEGF(165), starting 3 days after stroke, on functional neurological recovery, corticorubral plasticity and inflammatory brain responses in mice submitted to 30 min of middle cerebral artery occlusion. We herein show that the slowly progressive functional improvements of motor grip strength and coordination, which are induced by VEGF, are accompanied by enhanced sprouting of contralesional corticorubral fibres that branched off the pyramidal tract in order to cross the midline and innervate the ipsilesional parvocellular red nucleus. Infiltrates of CD45+ leukocytes were noticed in the ischemic striatum of vehicle-treated mice that closely corresponded to areas exhibiting Iba-1+ activated microglia. VEGF attenuated the CD45+ leukocyte infiltrates at 14 but not 30 days post ischemia and diminished the microglial activation. Notably, the VEGF-induced anti-inflammatory effect of VEGF was associated with a downregulation of a broad set of inflammatory cytokines and chemokines in both brain hemispheres. These data suggest a link between VEGF's immunosuppressive and plasticity-promoting actions that may be important for successful brain remodeling. Accordingly, growth factors with anti-inflammatory action may be promising therapeutics in the post-acute stroke phase.

An improved simple rat model for global cerebral ischaemia by induced cardiac arrest

OBJECTIVES Cerebral hypoxic-ischaemic injury following cardiac arrest is a devastating disease affecting thousands of patients each year. There is a complex interaction between post-resuscitation injury after whole-body ischaemia-reperfusion and cerebral damage which cannot be explored in in vitro systems only; there is a need for animal models. In this study, we describe and evaluate the feasibility and efficiency of our simple rodent cardiac arrest model. METHODS Ten wistar rats were subjected to 9 and 10 minutes of cardiac arrest. Cardiac arrest was introduced with a mixture of the short-acting beta-blocking drug esmolol and potassium chloride. RESULTS All animals could be resuscitated within 1 minute, and survived until day 5.General health score and neurobehavioural testing indicated substantial impairment after cardiac arrest, without differences between groups. Histological examination of the hippocampus CA1 segment, the most vulnerable segment of the cerebrum, demonstrated extensive damage in the cresyl violet staining, as well as in the Fluoro-Jade B staining and in the Iba-1 staining, indicating recruitment of microglia after the hypoxic-ischaemic event. Again, there were no differences between the 9- and 10-minute cardiac arrest groups. DISCUSSION We were able to establish a simple and reproducible 9- and 10-minute rodent cardiac arrest models with a well-defined no-flow-time. Extensive damage can be found in the hippocampus CA1 segment. The lack of difference between 9- and 10-minute cardiac arrest time in the neuropsychological, the open field test and the histological evaluations is mainly due to the small sample size.

106: Synthetic preimplantation factor (sPIF*) promotes neuroprotection by modulating PKA/PKC kinases

OBJECTIVE: Survivors of premature birth suffer from long term disabilities. Synthetic PreImplantation Factor (sPIF*) modulates inflammatory responses and reverses neuroinflammation. Proteinkinase A (PKA) and protein kinase C (PKC) are crucial signaling molecules. PKA up-regulates IL-10 and brain-derived neurotrophic factor (BDNF) expression, which exert neuroprotective effects. Anti-apoptotic phosphorylation of Bad is mediated by PKA. PKC phosphorylates GAP-43, a marker for neuronal plasticity and structural recovery. We explored sPIF protective role in neuronal (N2a) cells and in a rat model of encephalopathy of prematurity. *proprietary. STUDY DESIGN: Cells were subjected to LPS and treated with sPIF or scrambled sPIF. Neonatal rats (postnatal day 3: P3) were subjected to LPS, ligation of carotid artery, and hypoxia (8% O2, 65min; n¼ 30). sPIF (0.75mg/kg twice daily) was injected (P6-13) and brains harvested at P13. sPIF’s potential and mechanisms were evaluated using immunohistochemistry, ELISA, Western Blot, and qRT-PCR. Data were analyzed using two-tailed Student’s t-test. P<0.05 wasconsidered statistically significant. RESULTS: In vitro sPIF increased PKA/PKC activity in time dependent manner (Fig. 1A). sPIF induced higher IL-10, BDNF, and GAP-43 and lower CASP3, BAD, and TNF-a mRNA levels (Fig. 1B,C). sPIF increased pGap-43/Gap-43 and decreased pBad/Bad ratio while decreasing Bad (Fig. 1 D,E). In brain tissue sPIF treatment resulted in rescued neuronal number (NeuN positive cells) and reduced apoptosis (Casp-3 positive cells) with decreased glial (Iba-1 positive cells) activation (Fig. 2A,B). The Iba-1 morphology changed from predominantly amoeboid to ramified state. Additionally sPIF increased IL-10 mRNA levels (Fig. 2C) and pGap-43/Gap-43 ratio (Fig. 2D). CONCLUSION: sPIF modulates PKA/PKC pathways reducing apoptosis and inflammatory responses while increasing neuronal plasticity and survival. The identified PKA/PKC regulatory axis strengthens the potential of sPIF in reducing the burden of prematurity.

Automatic Counting of Microglial Cells in Healthy and Glaucomatous Mouse Retinas

Proliferation of microglial cells has been considered a sign of glial activation and a hallmark of ongoing neurodegenerative diseases. Microglia activation is analyzed in animal models of different eye diseases. Numerous retinal samples are required for each of these studies to obtain relevant data of statistical significance. Because manual quantification of microglial cells is time consuming, the aim of this study was develop an algorithm for automatic identification of retinal microglia. Two groups of adult male Swiss mice were used: age-matched controls (naïve, n = 6) and mice subjected to unilateral laser-induced ocular hypertension (lasered; n = 9). In the latter group, both hypertensive eyes and contralateral untreated retinas were analyzed. Retinal whole mounts were immunostained with anti Iba-1 for detecting microglial cell populations. A new algorithm was developed in MATLAB for microglial quantification; it enabled the quantification of microglial cells in the inner and outer plexiform layers and evaluates the area of the retina occupied by Iba-1+ microglia in the nerve fiber-ganglion cell layer. The automatic method was applied to a set of 6,000 images. To validate the algorithm, mouse retinas were evaluated both manually and computationally; the program correctly assessed the number of cells (Pearson correlation R = 0.94 and R = 0.98 for the inner and outer plexiform layers respectively). Statistically significant differences in glial cell number were found between naïve, lasered eyes and contralateral eyes (P<0.05, naïve versus contralateral eyes; P<0.001, naïve versus lasered eyes and contralateral versus lasered eyes). The algorithm developed is a reliable and fast tool that can evaluate the number of microglial cells in naïve mouse retinas and in retinas exhibiting proliferation. The implementation of this new automatic method can enable faster quantification of microglial cells in retinal pathologies.

Importância da interação entre a integrina Mac-1 dos leucócitos e a glicoproteína Iba das plaquetas para o recrutamento de leucócitos pelas plaquetas e para a resposta inflamatória à lesão vascular

OBJETIVO: Avaliar a importância da interação entre a integrina Mac-1 dos leucócitos (a Mb 2) e a glicoproteína (GP) Iba das plaquetas para o recrutamento de leucócitos após a lesão vascular e o efeito da neutralização da interação Mac-1-GPIba sobre a proliferação celular e a hiperplasia neointimal desencadeadas por lesão vascular. MÉTODOS: Um peptídeo denominado M2 ou anticorpo anti-M2 foi desenvolvido para bloquear a interação Mac-1-GPIba . Esse peptídeo foi injetado e comparado com anticorpo-controle em camundongos C57B1/6J submetidos a lesão vascular da artéria femoral com corda-guia. Um, cinco ou 28 dias após a lesão vascular, as artérias femorais foram retiradas para a realização de morfometria e imuno-histoquímica. RESULTADOS: O bloqueio da interação Mac-1-GPIba promoveu uma redução estatisticamente significativa do número de leucócitos na camada média no primeiro dia após a lesão vascular (controle: 7,9±5,0% do total de células versus anti-M2: 2,0±1,6%, p=0,021), bem como determinou uma diminuição estatisticamente significativa do acúmulo de leucócitos na neoíntima em cinco e 28 dias (controle: 42,3±12,9% versus anti-M2: 24,6±10,8%, p=0,047 e controle: 7,9±3,0% versus anti-M2: 3,3±1,3%, p=0,012; respectivamente). A proliferação celular na camada média do vaso em cinco dias pós-lesão foi reduzida com o bloqueio da interação Mac-1-GPIba (controle: 5,0±2,9% do total de células versus anti-M2: 1,8±0,5%; p=0,043), assim como houve diminuição significativa da proliferação celular na camada íntima do vaso em 28 dias (controle: 3,8±1,7% versus anti-M2: 2,0±1,2%; p=0,047). O bloqueio da interação Mac-1-GPIba também determinou uma redução estatisticamente significativa do espessamento intimal em 28 dias pós-lesão (controle: 10.395±3.549 µm² versus anti-M2: 4.561±4.915 µm²; p=0,012). CONCLUSÃO: O recrutamento de leucócitos após a lesão vascular é dependente da interação Mac-1-GPIba e a neutralização dessa interação inibe a proliferação celular e a formação neointimal.

IBA application for rooting of Eucalyptus benthamii Maiden and Cambage x Eucalyptus dunnii Maiden minicuttings

IBA application for rooting of Eucalyptus benthamii Maiden and Cambage x Eucalyptus dunnii Maiden minicuttings. Eucalyptus has great importance in the forestry sector and many advances in the area of improvement have been achieved with the advent of biotechnological techniques. However, some promising genotypes still do not have multiplication protocols with cloning techniques, such as minicutting. The study aimed to evaluate IBA concentrations for survival, rooting and vegetative vigor of E. benthamii x E. dunnii minicuttings, and determine the maximum technical efficiency dose. Ministumps H12, H19 and H20 clones were cultivated in a clonal minigarden under a semi-hydroponic system. For rooting, the rninicutting basal portion was plunged in hydro-alcoholic solutions, whose concentrations were: 0; 2,000; 4,000; 6,000 and 8,000 mg L(-1) of IBA. The experiment was conducted in a completely randomized design, with the factors consisting of three clones and five IBA concentrations, with five replications, containing 10 minicuttings per replication. IBA positively influenced the minicutting rooting processes, with differentiated behavior between the clones, with 30.32 to 55.45% rooting variation. The positives increments occurred until the highest IBA concentration for H12 and H19 clones. However, the range between 4,000 and 6,000 mg L(-1) of IBA treatments promoted the best rooting results for the H20 clone.

Luminosidade e IBA no enraizamento de microestacas de mirtileiro dos grupos Rabbiteye e Southern Highbush

O objetivo deste trabalho foi avaliar o efeito de diferentes concentrações do ácido indolbutírico (0 - controle; 500; 1.000; 1.500 e 2.000 mg L-1) no enraizamento de microestacas de mirtileiro de espécie e grupos distintos (cultivar O'Neal, Vaccinium sp., grupo Southern highbush e cv. Aliceblue, Vaccinium ashei Reade, grupo Rabbiteye), associado ao efeito da luminosidade na fase inicial do enraizamento das microestacas (14 dias de escuro ou luminosidade natural). Após o tratamento com IBA, as microestacas foram colocadas em bandejas de poliestireno expandido contendo como substrato perlita + Plantmax® (1:1) e mantidas em telado equipado com nebulização intermitente. Aos 60 dias após o estaqueamento, avaliaram-se a porcentagem de microestacas enraizadas, porcentagem de microestacas sobreviventes, número médio de raízes, comprimento médio de raízes e a porcentagem de formação de calo. Através dos resultados obtidos, concluiu-se que, para o enraizamento de microestacas de mirtileiro das cultivares O'Neal e Aliceblue, não é necessária a aplicação de IBA. A presença de luz na fase inicial do enraizamento não interfere na capacidade de enraizamento das microestacas. O tratamento com escuro na fase inicial do enraizamento reduz a formação de calos, promovendo diminuição da sobrevivência das microestacas e do percentual de enraizamento.

IBA e carboidratos no enraizamento de brotações procedentes de estacas radiciais de Rubus Spp.

Este trabalho objetivou verificar o efeito do ácido indol-3-butírico (IBA) e o teor de carboidratos na promoção do enraizamento em estacas de brotações de amoreira-preta. O experimento foi conduzido de junho a agosto de 2010, na UNESP de Botucatu - SP, sendo o delineamento em blocos casualizados, com seis concentrações de IBA e seis repetições, com a parcela constituída por 12 brotações. Os tratamentos constaram de seis concentrações de IBA, na forma de solução: T1= 0 mg L-1; T2= 250 mg L-1; T3= 500 mg L-1; T4= 1.000 mg L-1; T5= 2.000 mg L-1, e T6= 4.000 mg L-1 aplicados na base das brotações, durante dez segundos. Após 60 dias, foram avaliados: a porcentagem de enraizamento e o teor de carboidratos solúveis. As maiores concentrações de IBA inibiram o enraizamento das estacas de brotações. O aumento nos teores de açúcares da parte aérea com relação às raízes pode indicar que a parte aérea atuou como fonte de fotoassimilados e, dentre eles, açúcares solúveis, para promover o enraizamento das brotações.

IBA e carboidratos no enraizamento de brotações procedentes de estacas radiciais de Rubus Spp.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Influence of IBA and boric acid on rooting of stem-cuttings of Aloysia triphylla (L'Hérit) Britton

Aloysia triphylla (L'Hérit) Britton is a perennial and bushy plant, with simple, entire, lanceolate and whorl shaped leaves and originally from South America. It is used as medicinal plant in Brazil with stomatic and sedative properties. The employment of stem cuttings for propagation of pre-selected plants, acquired great importance, because it eliminates the juvenile phase of seedlings, which can be produced in a shorter period of time. The rooting of stem cuttings is stimulated by auxin and, boric acid supply is essential for growth and development of initial rootlets. This micronutrient is required 48 hours after plant segments have been placed into auxin solution and it can be supplied any time, including the seedling growth period. The experiment was carried out in the Department of Plant Production, UNESP-Botucatu-SP-Brazil, with stem-cuttings of Aloysia triphylla (L'Hérit) Britton, Verbenaceae obtained from the Medicinal and Aromatic Plant Garden. The aim of the work was to verify the influence of growth regulators and boric acid on stem cutting rooting of this species. The 15 cm-stem cuttings, without leaves, were submerged during 24 hs in the following solutions: water; 150 mg.L-1 of IBA; 150 mg.L-1 of IBA+ Boric acid; 250 mg.L-1 of IBA; 250 mg.L-1 of IBA + Boric acid. The statistical design was entirely randomized with 5 treatments and 3 replications, totalizing 15 plots with 10 stem cuttings each. They were planted on propylene trays with vermiculite and kept under spraying condition during twenty five days. The best results were observed in treatment 250 mg.L -1 of IBA+ Boric acid on number of roots, length of roots, rooting percentage, fresh and dry weight of leaves when compared with all other treatments. We can conclude that this treatment is the most suitable for stem cutting rooting of this species.

Enraizamento de estacas de pinheira (Annona squamosa L.), gravioleira (Annona muricata L.) e atemoeira (Annona squamosa L. x Annona cherimola L.) tratadas com ácido indolbutírico (IBA), ácido naftalenoacético (NAA) e bioestimulante

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Ácido indol-butírico, 2,6-di-hidroxiacetofenona e gene ARRO-1 no enraizamento de estacas de pessegueiro

Pós-graduação em Agronomia (Horticultura) - FCA

Clonagem do pessegueiro 'Aurora-1' e de portaenxertos de umezeiro

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)