Analysis of the SLIT2 protein in the rat cerebrospinal fluid during development

Analysis of the SLIT2 protein in the rat cerebrospinal fluid during development Abstract Introduction: The early ventricular system with it’s architecture surrounding midline structures and expansion to the brain cortex, and fluid flow plays dramatically a great role in the development of the central nervous system The cerebrospinal fluid contains distinct concentrations of variety of motogenes, mitogens, morphogenes, and many other essential growth factors and neurotrophines that are effectively programming the neural cell fates and thus brain architecture. SLIT2 a member of SLIT family of chemorepellents is famous for it’s role in midline crossing during axonal growth and arborization. Aim: Previous studies indicated the presence of SLIT2 mRNA in the neural cells and Choroid plexus during development. In the present study the presence of SLIT2 protein in the cerebrospinal fluid of the fourth ventricle of the brain is investigated and whether there is any perceivable fluctuations in the concentration of this protein. Materials & Methods: Based on ethics, over 1000 rat fetuses during embryonic days 16‐20 were applied to cerebrospinal fluid extraction from the cisterna magna region of the brain with glassy micropipettes. The pure cerebrospinal fluid specimens subsequently were centrifuged (1500 rpm for 3‐5 min) and stored in ‐80 degree Celsius for 2‐3 months. The sandwich ELISA protein assay was applied respectively for both technical and experimental repeats. The repeats were 3‐8 times according to the restrictions of specimen’s volume which were due to fetal sizes. Results: The ELISA assay indicated that there are noticeable concentrations of SLIT2 during Embryonic days 16, 17, and 20. Besides there are countable concentrations of SLIT2 during embryonic days 18, and 19. There was a rise from E16 to E17, almost the concentration was doubled. From E17 to E18 there was a dramatic fall, so close to unmeasurable amount . A gradual rise from E18 to E19 was observed. From E19 to E20 a sharp rise almost at the same concentration of E17 was perceived. Conclusions: The ELISA assay clearly revealed the presence of SLIT2 protein in the cerebrospinal fluid of the fourth ventricle of the brain during embryonic days 16‐ 20 of fetal development. Furthermore, there were obviously perceivable fluctuations in the concentrations measured during this assay both in experimental and technical assays that suggest there should be a pattern of protein expression in the fourth ventricle that could be due to the role of SLIT2 protein in the different developmental stages. Experimental repeats Technical repeats Comparing two assays Summar Winter Consenteation ngr/m*10l 60 50 40 30 20 10 0 E16 E17 E18 E19 E20 Embryonic day Mid of assays 60٫0000 50٫0000 40٫0000 30٫0000 20٫0000 10٫0000 0٫0000 E16 E17 E18 Embryonic day E19 E20