We have analyzed the immunohistochemical expression of chondroitin sulfate proteoglycan (CSPG), fibronectin (FN), laminin (LN), tenascin (TN), and glial fibrillary acidic protein (GFAP) along the anterior commissure (AC) of hamster embryos (n=175; from embryonic day (E)12 to E16). Frozen sections were cut at different planes from embryonic brains between E12 and E16, treated for immunohistochemistry, and observed under epifluorescence microscopy. During the pre-crossing stage (E12-E13), CSPG was expressed as a sagittal stratum between the interhemispheric fissure and the prospective AC region. TN appeared rostral to the third ventricle and along the medial subventricular zone of the lateral ventricles. LN and FN both presented a faint expression, and GFAP was not detected. Although AC axons started crossing the midline region (E13.5-E14), CSPG, FN, LN, and, much less intensely, GFAP circumscribed the AC bundle, forming a tunnel through which AC fibers elongate. TN was no longer seen at the midplane but remained visible laterally. During the post-crossing stage (E14.5-E16), CSPG and TN were no longer seen at the midline, although both could be observed between the AC limbs, seeming to form boundaries for AC lateral growth. LN and FN were then absent near the AC bundle. During this late stage, GFAP expression became most intense, forming a distinct tunnel around the AC. We have shown that the expression of extracellular matrix molecules and GFAP follow a time- and space-regulated course related to AC development, plausibly representing influential factors for growth and guidance of commissural fibers.

This study aimed to investigate the antitumor and immunomodulatory properties of the flavonoid apigenin (5,7,4'-trihydroxyflavone), which was extracted from Croton betulaster Mull, in glioma cell culture using the high-proliferative rat C6 glioma cell line as a model. Apigenin was found to have the ability to reduce the viability and proliferation of C6 cells in a time-dependent and dose-dependent manner, with an IC50 of 22.8 µmol/l, 40 times lower than that of temozolomide (1000 µmol/l), after 72 h of apigenin treatment. Even after C6 cells were treated with apigenin for 48 h, high proportions of C6 cells entered apoptosis (39.56%) and autophagy (22%) as shown by flow cytometry using annexin V/propidium iodide and acridine orange staining, respectively. In addition, the flavonoid apigenin induced cell accumulation in the G0/G1 phase of the cell cycle and inhibited glioma cell migration efficiently. Moreover, apigenin induced astroglial differentiation and morphological changes in C6 cells, characterized by increased expression of glial fibrillary acidic protein and decreased expression of nestin protein, a typical marker of neuronal precursors. The immunomodulating effects of apigenin were also characterized by a change in the inflammatory profile as evidenced by a significant decrease in interleukin-10 and tumor necrosis factor production and increased nitric oxide levels. Because apigenin can induce differentiation, apoptosis, and autophagy, can alter the profile of cytokines involved in regulating the immune response, and can reduce the survival, growth, proliferation, and migration of C6 cells, this flavonoid may be considered a potential antitumor drug for the adjuvant treatment of malignant gliomas.

We have analyzed the immunohistochemical expression of chondroitin sulfate proteoglycan (CSPG), fibronectin (FN), laminin (LN), tenascin (TN), and glial fibrillary acidic protein (GFAP) along the anterior commissure (AC) of hamster embryos (n=175; from embryonic day (E)12 to E16). Frozen sections were cut at different planes from embryonic brains between E12 and E16, treated for immunohistochemistry, and observed under epifluorescence microscopy. During the pre-crossing stage (E12-E13), CSPG was expressed as a sagittal stratum between the interhemispheric fissure and the prospective AC region. TN appeared rostral to the third ventricle and along the medial subventricular zone of the lateral ventricles. LN and FN both presented a faint expression, and GFAP was not detected. Although AC axons started crossing the midline region (E13.5-E14), CSPG, FN, LN, and, much less intensely, GFAP circumscribed the AC bundle, forming a tunnel through which AC fibers elongate. TN was no longer seen at the midplane but remained visible laterally. During the post-crossing stage (E14.5-E16), CSPG and TN were no longer seen at the midline, although both could be observed between the AC limbs, seeming to form boundaries for AC lateral growth. LN and FN were then absent near the AC bundle. During this late stage, GFAP expression became most intense, forming a distinct tunnel around the AC. We have shown that the expression of extracellular matrix molecules and GFAP follow a time- and space-regulated course related to AC development, plausibly representing influential factors for growth and guidance of commissural fibers.

The flavonoid apigenin, extracted from the Brazilian plant Croton betulaster Müll. has demonstrated the ability to inhibit proliferation, induce differentiation, and modify the inflammatory profile of glioma cells. The aim of the present study was to evaluate the effect of apigenin on chemotaxis and regulation of inflammatory cytokines of microglia cells and these impacts on glioma cell growth. In cultures of isolated rat microglia, it was observed that apigenin induced changes in Iba1‐positive cells to an ameboid phenotype, associated to an increase in the expression of the activated M1 profile marker OX‐42 and iNOS and a reduction in the expression of the M2 profile marker CD206. Besides, apigenin modulated the tumor necrosis factor and IL‐10 release by microglia. Treatment of C6 glioma cells with conditioned medium of microglia treated with apigenin‐induced reduction of tumor migration and viability, associated with significant reduction in IL‐6 levels. On the other hand, treatment of C6 cells with apigenin‐induced microglia chemotaxis to glioma in vitro. Moreover, apigenin treatment of microglia/C6 co‐cultures induced preferentially reduction in the viability of C6 cells and increased microglia‐activated phenotype, associated with a change in the balance of TNF/IL‐10 levels. Together, these results demonstrated that the flavonoid apigenin restores the immune profile of microglia against glioma cells.