霍格兰营养液配方

霍格兰营养液配方 COMMENTS FOR THE AUTHOR: Reviewer #1: The manuscript CEMN-D-12-01363 by Chen and collaborators describes the expression and distribution of the transcription factor FOXO3a and the kinase inhibitor p27kip1 in the retina of the DBA/2J mouse relative o the C57/BL6 mice. The manuscript contains some original observations. SUMMARY: While Foxo3a inhibits cell cycle progression via control of p27kip1 (cyclin kinase inhibitor) during the G1-S phase transition in various cell lines, the authors believe these two genes/gene products are worthy candidates for study in differentiated neurons and glia in a mouse model for glaucoma. They looked for the spatial and temporal distributions in the retinas of the DBA/2J mice (D2) grouped by factors such as age, IOP, slit lamp and ophthalmoscope inspection to delineate non-glaucomatous, pre-glaucomatous and glaucomatous animal groups. The authors used light microscopy, RT-PCR, and immunocytochemistry to look at the spatial and temporal distributions and expression levels of foxo3a and p27kip1 and made use of TUNEL and antibodies to activated caspase-3 as measures of cell death. Their results indicated that - Westerns: foxo3a and p27kip proteins were reduced in time/condition relative to control mice retina; RT-PCR: p27kip mRNA may also be reduced with time/condition. Immuno-histology was used to show that - foxo3a and p27kip are diffusely expressed in retina of D2 and B6 mice and decreased in Muller (GS+) and astrocytes (GFAP+). However, foxo3a increased in RGCs (neuN+) of D2 mice and activated caspase 3 was evident in RGCs (NeuN+) of D2 mice. TUNEL was evident in RGC layer of D2 mice The authors concluded that - foxo3a and p27kip1 are involved in neural cell loss in D2 mice CRITIQUE: INTRODUCTION: The experimental plan to group the animals into 3 groups is a good idea, although, it has been shown previously that there are changes the expression of some genes that occur prior to the full blown glaucomatous condition. In this case, it appears that FOXO3a and p27kip1 may be somewhat late and perhaps downstream to the primary signaling events leading to loss of vision. "ubiquitously" seems to be redundant in first sentence of second paragraph. The listed citations do not give appropriate credit to many important studies. For example: 1) The introduction to this paper needs additional references for the second sentence in the 1st paragraph, especially for each of the mechanisms suggested as primary causes of RGC cell death. The author should add excitotoxicity as one of the postulated mechanisms here - especially as foxo3a has been implicated in apoptosis. 2) Paragraph 4 of the introduction - the authors may want to cite the 2001 Nobel laureates for their discoveries of the role of cyclins and CDKs in cell cycle progression. 3) The authors must cite Ophthalmol Eye Dis. 2010 March 11; 1: 23-41, a microarray study where up-regulation of GFAP (Muller cells and astrocytes) and Iba1 (microglia) were shown and where a CDI was also shown to be increased in the DBA/2J mouse retina. At the very least this should be in the last paragraph of the introduction, and probably, also used in the discussion. 4) The authors should also cite Invest Ophthalmol Vis Sci 2006;47:977-85 another important gene expression study. This should also go, at least in the last paragraph of the introduction. METHODS: Can the authors state how they determined an illumination level of 50-60 lux for housing the animals? Just curious. I would like to see a table for the 3 DBA/2J animal groups with the various factors used to assign animals to this or that group. This might be helpful to investigators who use these animals. Catalogue numbers should be used to identify the antibodies that were used. RESULTS: In second and fifth lines of the first paragraph, what is meant by the term "control group" here? Are these B6 or are they D2- non glaucomatous mice? You also use the term "control" for no-primary controls, for example, in the figure legends. So always be specific. The last sentence of the first paragraph states that neurodegeneration induced a decrease in foxo3a and p27kip1. The term "induced" may be inappropriate here. The second paragraph speaks of a decrease in immuno-staining. This is difficult to discern, perhaps due to the use of cresyl violet as a counterstain. It might be better to not use a nuclear counterstain. I cannot see the translocation from nucleus to cytoplasm. Again this might have been possible without the dark blue counterstain. I cannot see much overlap between foxo or p27 with the Muller cell glutamine synthase in figure3. While the author has boxed a possible example, there are many instances where the labeling is either red or green. There are hints of foxo and p27 in the middle layer of the INL and in the OPL, but the figure could be improved. There is some evidence in Figure 4 for double labeled cells containing both markers for GFAP and Foxo but all-GFAP or all-Foxo labeled cells are also to be seen. Can the authors explain this? The statement indicating a cause and effect relationship between FOXO and p27 may be misplaced, at least in this part of the results section. There does appear to be a correlation between the relative abundance of FOXO and p27 in the retina as judged from western blots of the whole retina, but you have also made a case for increased abundance of FOXO in RGCs. Thus, it is not clear that your argument is consistent at the cellular level, or at least, it is not made clear to this reviewer. It is not clear that figure 8 adds anything that has not already been reported and the data are not that convincing anyway. It might have been useful to show the presence of cleaved caspase or TUNEL in double labeled cells containing FOXO or p27 because the direct connection between FOXo and p27 with cell death is somewhat tenuous in this manuscript. DISCUSSION: Much is lost in translation and this is particularly true of the discussion section where misleading statements such as "The present data reveal that down-regulation of p27kip1 through FOXO transcriptional inhibition was " and "our results demonstrated that Foxo3a as a associated with ---- positive regulator of p27kip1 was associated with ---" can be found. There is little integration with the existing data on the DBA/2J mouse or any other animal model of glaucoma. Reviewer #2: In this study the authors show that the transcription factor FOXO3a and its downstream gene p27kip1 are decreased in glaucomatous DBA/2J retinas. The decreased expression of these proteins was found in the Müller cells and astrocytes. The authors also found increased expression of FOXO3a in RGCs from glaucomatous retinas. The latter finding seems to be correlated with the apoptosis of RGCs, observed with TUNEL and Caspase3 staining in glaucomatous retinas. Major points: 1) The authors were able to show that FOXO3a and p27kip1 expression levels are changing in glaucomatous retinas, however the study lacks a direct connection between their findings and the cellular processes in which these proteins are involved. Downregulation of FOXO3a and p27kip1 have been proposed to be involved in cell proliferation, while upregulation of FOXO3a in neurons is associated with apoptosis. It will be interesting if the authors are able to establish the connection between the expression of these proteins and the cellular events mentioned above. A previous study has shown the involvement of the PTEN-Akt-FOXO3a pathway in neuronal apoptosis in brain development after hypoxia-ischemia (Li et al., J Cereb Blood Flow Metab, 2009). In this study, the expression of Bim, an apoptosis related protein that is downstream of FOXO3a, is increased as a response of FOXO3a nuclear translocation. The authors could demonstrate the link between FOXO3a and apoptosis by showing dephosphorylation in the PTEN-Akt-FOXO3a pathway and the consequent increased levels of Bim in glaucomatous RGCs. In this way the authors might propose a possible mechanism of apoptosis that could be targeted therapeutically in glaucoma. 2) It is necessary to include an optic nerve assessment of degeneration for the establishment of glaucoma in these mice. The use of an axonal antibody in the optic nerve could be a good alternative.