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The aim of this study was to evaluate the effects of neuropsychopedagogical intervention (INPp) on the academic performance of students with learning difficulties (LD). A total of 4,184 children aged between 6 and 8 years (±7.23) of both genders participated in the present study. They were divided into four groups (A1 –1151 children who did not present LD and who underwent INPp; A2 – 1151 children without LD who did not undergo INPp; B1 - 942 children who presented AD and who underwent INPp; B2 - 942 children with LD and who did not undergo INPp). The INPP was developed through the systematic practice of 04 playful activities, in three weekly sessions, lasting 45 minutes each for 18 sessions (±2 months). In order to evaluate the effects of the INPP, the participants were submitted to the following protocols: a) Dual Choice Mental Processing Test (DMCPT) ; b) Rapid Automatized Naming (RAN); c) Assessment of academic performance in Portuguese and Mathematics. All protocols were approved by the UFRJ ethics committee (opinion no. 517,483) and carried out in the school environment. Our results show that children in group A1 when evaluated after INPp presented an 18% reduction (p

Bevacizumab, an anti-vascular endothelial growth factor (VEGF) agent, is widely used in the treatment of retinal vascular diseases. However, due to the essential role Muller cell derived-VEGF plays in the maintenance of retinal neurons and glial cells, cell viability is likely to be affected by VEGF inhibition. We therefore evaluated the effect of bevacizumab-induced VEGF inhibition on Muller cells (MIO-M1) in vitro. MIO-M1 cells were cultured for 12 or 24 h in media containing bevacizumab at 0.25 or 0.5 mg/mL. Controls were cultured in medium only. Cell viability was determined with the trypan blue exclusion test and MTT assay. Caspase-3, beclin-1, glial fibrillary acidic protein (GFAP) and vimentin content were quantified by immunohistochemistry. Gene expression was evaluated by real-time quantitative PCR. Treatment with bevacizumab did not reduce MIO-M1 cell viability, but increased metabolic activity at 24 h (0.5 mg/mL) and induced apoptosis and autophagy, as shown by the increased caspase-3 levels at 12 h (0.25 and 0.5 mg/mL) and the increased beclin levels at 24 h (0.5 mg/mL). Caspase-3 mRNA was upregulated at 12 h and downregulated at 24 h in cells treated with bevacizumab at 0.25 mg/mL. Bevacizumab treatment was also associated with structural protein abnormalities, with decreased GFAP and vimentin content and upregulated GFAP and vimentin mRNA expression. Although bevacizumab did not significantly affect MIO-M1 cell viability, it led to metabolic and molecular changes (apoptosis, autophagy and structural abnormalities) suggestive of significant cellular toxicity.

The retinotectal projection of rodents presents a precise retinotopic organization that develops, from diffuse connections, from the day of birth to post-natal day 10. Previous data had demonstrated that these projections undergo reorganization after retinal lesions, nerve crush and monocular enucleation. The axonal growth seems to be directly related to growth-associated protein-43 (GAP-43) expression, a protein predominantly located in growth cones, which is regulated throughout development. GAP-43 is presented both under non-phosphorylated and phosphorylated (pGAP-43) forms. The phosphorylated form, has been associated to axon growth via polymerization of F-actin, and synaptic enhancement through neurotransmitter release facilitation. Herein we investigated the spatio-temporal expression of GAP-43 in the rat superior colliculus during normal development and after monocular enucleation in different stages of development. Lister Hooded rats ranging from post-natal day 0 to 70 were used for ontogeny studies. Another group of animals were submitted to monocular enucleation at post-natal day 10 (PND10) or PND21. After different survival-times, the animals were sacrificed and the brains processed for either immunohistochemistry or western blotting analysis. Our data show that GAP-43 is expressed in retinotectal axons in early stages of development but remains present in adulthood. Moreover, monocular enucleation leads to an increase in pGAP-43 expression in the deafferented colliculus. Taken together these results suggest a role for pGAP-43 in retinotectal morphological plasticity observed both during normal development and after monocular enucleation.

PURPOSE Extracellular matrix (ECM) and cellular membrane proteoglycans (PGs) play important roles in neural differentiation and cell adhesion. Vascular endothelial growth factor, an important signal protein in vascular and retinal neural cell development, is retained in the ECM due to its high affinity for PG. Bevacizumab, an anti-VEGF agent, has been extensively used for treating retinal diseases in adult and newborn patients, although its effect on the developing retina remains largely unknown. The purpose of this study was to investigate the effect of bevacizumab on neurocan, phosphacan, and syndecan-3 PG levels in newborn rat retina. METHODS Retinal explants of sixty 2-day-old Lister hooded rats were obtained after eye enucleation and maintained in culture media with or without bevacizumab for 48 hours. Immunohistochemical staining was assessed against neurocan, phosphacan, and syndecan-3. Proteoglycan content was quantified based on the intensity of immunohistochemical labeling. Gene expressions were quantified by real-time reverse-transcription polymerase chain reaction. The results from the treatment and control groups were compared. RESULTS No significant difference in the staining intensity and mRNA expression of phosphacan and syndecan-3 was observed between the groups. However, a significant decrease in neurocan content and mRNA expression was observed in bevacizumab-treated retinal explants compared with controls. CONCLUSIONS Bevacizumab did not affect phosphacan and syndecan-3 levels but decreased neurocan content and gene expression. Therefore, it may interfere with early postnatal retinal cell differentiation. Although further studies are necessary to confirm our findings, we suggest anti-VEGF agents be used with caution in developing retinal tissue.

PURPOSE Vascular endothelial growth factor (VEGF) is an important signal protein in vertebrate nervous development, promoting neurogenesis, neuronal patterning, and glial cell growth. Bevacizumab, an anti-VEGF agent, has been extensively used for controlling pathological retinal neovascularization in adult and newborn patients, although its effect on the developing retina remains largely unknown. The purpose of this study was to investigate the effect of bevacizumab on cell death, proliferation, and differentiation in newborn rat retina. METHODS Retinal explants of sixty 2-day-old Lister hooded rats were obtained after eye enucleation and maintained in culture media with or without bevacizumab for 2 days. Immunohistochemical staining was assessed against proliferating cell nuclear antigen (PCNA, to detect cell proliferation); caspase-3 and beclin-1 (to investigate cell death); and vimentin and glial fibrillary acidic protein (GFAP, markers of glial cells). Gene expressions were quantified by real-time reverse-transcription polymerase chain reaction. Results from treatment and control groups were compared. RESULTS No significant difference in the staining intensity (on immunohistochemistry) of PCNA, caspase-3, beclin-1, and GFAP, or in the levels of PCNA, caspase-3, beclin-1, and vimentin mRNA was observed between the groups. However, a significant increase in vimentin levels and a significant decrease in GFAP mRNA expression were observed in bevacizumab-treated retinal explants compared with controls. CONCLUSIONS Bevacizumab did not affect cell death or proliferation in early developing rat retina but appeared to interfere with glial cell maturation by increasing vimentin levels and downregulating GFAP gene expression. Thus, we suggest anti-VEGF agents be used with caution in developing retinal tissue.

Purpose VEGF is an important in nervous development, promoting neurogenesis and glial cell growth. Bevacizumab (BVZ), an anti-VEGF agent, has been extensively used for controlling pathological retinal neovascularization in adult and newborn patients, although its effect on the developing retina remains largely unknown. In this study we investigate BVZ effect on proteoglycans and on cell death, proliferation and differentiation in newborn rat retina. Methods Retinal explants of sixty 2-day-old Lister hooded rats were maintained in culture media with or without BVZ for 2 days. Immunohistochemical staining was assessed against proliferating cell nuclear antigen (PCNA), caspase-3 and beclin-1 and vimentin and glial fibrillary acidic protein (GFAP). Gene expressions for PCNA, caspase-3, beclin-1, vimentin, GFAP and for proteoglycans were quantified by real-time reverse-transcription polymerase chain reaction. Results from treatment and control groups were compared Results No significant difference in staining intensity of PCNA, caspase-3, beclin-1 and GFAP or in the mRNA levels of PCNA, caspase-3, beclin-1, vimentin, neurocan and phosphacan was observed. However, a significant increase in vimentin levels, decrease in GFAP and in syndecan-3 mRNA expression were observed in BVZ-treated retinas compared to controls. Conclusion Bevacizumab did not affect cell death or proliferation in early developing rat retina but appeared to interfere with glial cell maturation (by interfering with vimentin and GFAP). It can also alter syndecan-3 gene expression. Thus, we suggest anti-VEGF agents be used with caution in developing retinal tissue.

Retinal dystrophies involve extensive photoreceptor apoptosis. Neuroprotective effects of insulin-like growth factor (IGF)-1 have been demonstrated in various tissues, including the retina. The aim of this study was to investigate: (i) the action of IGF-1 upon selective photoreceptor death induced by okadaic acid (OA); and (ii) signaling pathways related to both OA-induced cell death and IGF-1 neuroprotective effect. Retinal explants were incubated with 5nM OA, a protein phosphatase type 1 and type 2A inhibitor, which induces cell death detected by the identification of pyknotic morphology of photoreceptors immunostained for rhodopsin. OA increased both the number of pyknotic Rho 4D2(+) profiles, and Ca(2+) influx, measured through the incorporation of (45)CaCl(2), in a dose- and time-dependent way, while treatment with 10ng/mL IGF-1 abrogated both effects. Treatment with phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C, modulated OA effects, indicating the involvement of PKC. Furthermore, either 10microM chelerythrine chloride, an inhibitor of PKC, or 10microM nifedipine, a L-voltage-sensitive Ca(2+) channel blocker, inhibited both Ca(2+) influx and cell death induced by OA. The data show that okadaic acid induces rod photoreceptor cell death in retinal tissue through activation of PKC and ensuing Ca(2+) influx through L-type Ca(2+) channels, which is counteracted by a neuroprotective effect of IGF-1.

Cytokines are a heterogeneous group of molecules that have been associated with several functions in the nervous system, such as survival and differentiation of neuronal and glial cells. In the present study, we demonstrated that conditioned medium from spleen cells activated with concanavalin A increased neuritogenesis and survival of retinal cells, as measured by biochemical and morphological criteria. Our data showed that conditioned medium induced a five-fold increase in the amount of protein after 120 h in vitro. This effect was not inhibited by the blockade of voltage-dependent L-type calcium channels with 5.0 microM nifedipine. However, the use of an intracellular calcium chelator (15.0 microM BAPTA-AM) inhibited this effect. Our results support the idea that factors secreted by activated lymphocytes, such as cytokines, can modulate the maintenance and the differentiation of rat retinal cells in vitro, indicating a possible role of these molecules in the development of retinal cells, as well as in its protection against pathological conditions.