Computational protocol: Neuroprotection and neuroregeneration of retinal ganglion cells after intravitreal carbon monoxide release

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Protocol publication

[…] IRI activates glial cells and their reactivity, inducing both neuroprotective and neurodegenerative modes of action. Two types of macroglial cells can be found in the mammalian retina, astrocytes and Müller cells; they express GFAP as an intermediate filament protein. Although Müller cells express little or no GFAP in healthy rat retinae, they show increased GFAP expression after retinal injuries, including IRI []. To quantify GFAP-positive cell migration out of retinal explants seven days after IRI, photographs of the entire explants were taken with a fluorescence microscope (Observer Z.1; Carl Zeiss, Jena, Germany) using the stack and tile function and composed into mosaics. The total area of GFAP-positive cells which passed the explant margin was calculated into pixels with ImageJ using the threshold tool. The entire area of the trepanned explant was also evaluated into pixels to compensate for fluctuations in their size. The pixels of the GFAP-positive area were divided by the area of the explant to achieve the so-called GFAP-quotient. Results are expressed as the median GFAP-quotient in box plots for each treatment group seven days after IRI (n = 7).Class III beta-tubulin (beta-III tubulin) is a microtubule element of the tubulin family, which is primarily expressed in neurons and might be involved in neurogenesis and axon guidance and maintenance. Normally, glial cells do not express beta-III tubulin, although the coexpression of this marker in fetal or reactive astrocytes has been reported [, ]. Double-immunolabeled cells (GFAP- and beta-III tubulin-positive cells) were detectable after culturing and counted circumferentially using ZEN-blue 2010 software. Results are expressed as median absolute numbers in box plots for each treatment group seven days after IRI (n = 7). [...] Data of RGC-quantification and protein expression were analyzed as follows: A One-way-ANOVA with multiple comparison analysis (Tukey Contrasts) was used to compare the ischemic eyes and the intravitreal treatment. A paired t-test was used for comparisons between right and left eyes to take into account the dependent nature of these values. Data of mRNA were analyzed using a computerized statistical program (SigmaPlot Version 11.0, Systat Software Inc., San Jose, CA, USA). The results are presented as means (±SD) after normal distribution of the data had been verified. One-way ANOVA for repeated measurements was used for between-group comparisons with a post hoc Holm-Sidak test. P<0.05 was considered statistically significant. Axonal resprouting and glial migration out of the retinal explants were analyzed with the R-System. Results are presented in box plots as a median and quartiles. The Kruskal-Wallis-test and ANOVA with post hoc analysis were used to assess statistical significance. Again, P-values below 0.05 were considered statistically significant. The unoperated right eyes of experimental animals were used for comparison. […]

Pipeline specifications

Software tools ImageJ, SigmaPlot
Applications Miscellaneous, Microscopic phenotype analysis
Organisms Rattus norvegicus
Chemicals Carbon Monoxide