Water Extract of Mixed Mushroom Mycelia Grown on a Solid Barley Medium Is Protective against Experimental Focal Cerebral Ischemia

Round 1

Reviewer 1 Report

In this study, it was demonstrated the neuroprotective effects of mixed mushroom mycelia (MMM) against experimental fCI, leading the way to a possible supplement to the diet as a preventive strategy against fCI.

Introduction

In the Introduction section better explain why [Ca²⁺]_i increases after ischemic insult. See Dirnagl et al., Trends Neurosci. (1999) 22, 391–397.

Materials and Methods

1. In order to simulate the damage exterted by the glutammate during ischemic excitotoxic phase, the authors added glutammate to cells in colture. Why did they not perform an Oxigen and Glucose Deprivation (OGD) experiment? That is a simil-ischemic insult in vitro?
2. Why the authors chose 2 h after tMCAo for ROS detection assay and Western Blot analysis?
3. Animals number for ROS detection was very low (n=2), how could it reach some statistical relevance?
4. Specify what “NDS” indicates (line 235, Materials and Methods section).

Results

1. Add the statistical analysis used in the figure legends.
2. To study the anti-apoptotic effects of w-MMM in ischemia, the authors conducted a histological analysis with C-V stainig to individuate neurons with picnotic nucleus, a TUNEL-staining e then an immunohistochemical assay for Cleaved caspase-3. It could be interesting to show a qualitative immages of each experimental groups of a doble-immunofluorescence staining with anti-Neun and Cleaved caspase-3 antibodies, in order to visualize the apoptotic neurons.
3. Indicate where the photos of Figure 6 and 7 were captured. Cortical or striatal areas? 
4. Increase the resolution of the photo in Figure 3 A-B-E; 6 A-B-C; 7 A.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Jeong et al. Water extract of mixed mushroom mycelia grown on a solid barley medium is protective against experimental focal cerebral ischemia

In the present manuscript, Jeong and co-workers investigate the role of mushroom extracts on PC-12 cells and on rats undergoing an experimental stroke model. The authors show that the water fraction of their mixed mushroom mycelia (MMM) has low toxicity and protects PC-12 cells from reactive oxygen species and apoptosis. Likewise, a pre-treatment of rats with the MMM water fraction reduced infarct sizes following 90 min tMCAO.

The topic of the study is of interest and the paper is well written. However, some concerns should be addressed before this manuscript can be accepted for publication:

• Please provide order numbers or clone names for the antibodies used in this study, so that it is clear what has been used for this manuscript. Have isotype controls been used (I hope so). Please mention this and provide data in the supplemental material.
• What is the rationale for using SEM instead of the more appropriate SD-values? The authors should correct this (or even better blot median and IQR including the individual values of each sample). Likewise, n-numbers and nature of biological samples (cells, runs, rats) as well as the statistical tests should be mentioned in the figure legends. The authors should also check for normal distribution and adept their statistical tests for those cases that are not normally distributed.
• The preparation of the extracts appears to refer to a yet unpublished study to which I did not have access, making it hard to judge this part of the manuscript (albeit I do not really think that this is a concern).
• wMMM-L and wMMM-H should be explained at first appearance in the result text as well as in the relevant figure legends and not only in the method section.
• For the immunofluorescence images in Fig. 6 and Fig. 7 it would be helpful if a scheme of the brain indicating which region was analysed was provided.
• Providing the very strong protective effect of wMMM on the neurological outcome after focal cerebral ischemia, it would be very interesting to see whether wMMM-H would have some protective effect if administered in an therapeutical setting (giving it after ischemia – perhaps by injecting it).

Author Response

Please see the attachment.

Author Response File: Author Response.pdf