Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

An article by Sato and Schluter entitled „Effects of voluntary running wheel activity and hypertension 2 on the brain of female spontaneously hypertensive rats (SHR)" investigated the molecular targets that link hypertension to Alzheimer’s disease and those that can be affected by physical activity in female hypertensive rats. The article is well written and organised, and brings new and potentially clinically applicable results.

I have a few questions and comments about the manuscript:

1. Alzheimer's disease typically occurs in older age. In the study, rats aged 6 weeks to 7.5 months were used, which are still relatively young rats. Why didn't you choose older rats? Would you expect similar results when using older rats? Please discuss this issue in the Discussion.

2. No behavioral tests, such as the open field test to test locomotor activity, or other tests to monitor memory improvement, were performed in the work. If you have conducted such studies, please include them in the manuscript to strengthen the conclusions of your study.

3. Six Wis, 21 SHR, and 15 SHR-R were used in the study. However, the methods do not state the number of rats used for individual determinations. Please provide the exact number of rats used for individual analyses in each method or figure legends.

4. More technical information needs to be provided in the Methods. Provide more details on how the rats were housed (temperature, humidity, light regimen), how many rats were in one cage, what diet they received, whether they were fed ad libitum, how you controlled the voluntary running of individual rats, and wheel size. Provide information on where the rats were obtained/purchased from.

5. Rat genes are conventionally written in italics, and only the first letter is capitalized (i.e., Sod, Cat...) instead of SOD, CAT, etc. Please correct the gene abbreviations in the text and images.

At the end of the text, delete the sections Appendix A and B.

Author Response

We thank the reviewer for critical evaluation of our manuscript and study. Please find enclosed a point-by-point response to your suggestions.

You wrote: An article by Sato and Schluter entitled „Effects of voluntary running wheel activity and hypertension 2 on the brain of female spontaneously hypertensive rats (SHR)" investigated the molecular targets that link hypertension to Alzheimer’s disease and those that can be affected by physical activity in female hypertensive rats. The article is well written and organised, and brings new and potentially clinically applicable results.

Response: We thank the reviewer for this summary remark.

You wrote: I have a few questions and comments about the manuscript:

1. Alzheimer's disease typically occurs in older age. In the study, rats aged 6 weeks to 7.5 months were used, which are still relatively young rats. Why didn't you choose older rats? Would you expect similar results when using older rats? Please discuss this issue in the Discussion.

Response: Alzheimer’s disease is diagnosed typically in older people. However, at the time when it is diagnosed it is the endpoint of a disease progression that has probably started before and not at the time of diagnosis. Our study aimed at identifying molecular mechanisms that are probably involved in this process. As midlife hypertension has been associated with Alzheimer’s Disease (ref. 1 and 2) we considered adult hypertensive rats as a suitable model to identify potential mechanisms leading to Alzheimer’s disease. Therefore, we did not use older SHRs as they have already developed impaired memory (ref. 11).

You wrote: 2. No behavioral tests, such as the open field test to test locomotor activity, or other tests to monitor memory improvement, were performed in the work. If you have conducted such studies, please include them in the manuscript to strengthen the conclusions of your study.

Response: Memory testing of older SHRs (14-15 months) has been performed before (ref. 11) and was not done in our study. As we focused on mechanisms that may lead to Alzheimer’s disease and not necessarily pop up only at the time of diagnosis, we do not expect that these tests will identify phenotypically relevant singes at that time. In progression of the current study we aimed to use UCP2 knockout rats (that we have in our Lab) and want to address the questions whether UCP2 downregulation alone is sufficient to induce similar memory defects as mentioned before in SHR during ageing. However, we this type of experiment is the conclusion of our current study and not in the scope of the current investigation.

You wrote: 3. Six Wis, 21 SHR, and 15 SHR-R were used in the study. However, the methods do not state the number of rats used for individual determinations. Please provide the exact number of rats used for individual analyses in each method or figure legends.

Response: We have carefully checked all figure legends and included the exact number of rats used per experiment where it was missing before. In general we used all rats described in the study protocol, but we had limitations in blood pressure measurements (restriction requested by the ethical bord) and of course we had to select randomly rats for WBs (as the number of lanes is limited) and for histology. No animals died during the experiments or were not further considered for any other reason.

You wrote: 4. More technical information needs to be provided in the Methods. Provide more details on how the rats were housed (temperature, humidity, light regimen), how many rats were in one cage, what diet they received, whether they were fed ad libitum, how you controlled the voluntary running of individual rats, and wheel size. Provide information on where the rats were obtained/purchased from.

Response: We added the information about housing conditions: Temperature was 21-22 ^oC, humidity 60%, light regime was 12-12 h (dark-light cycle), two rats were housed in cages (length 595 mm, bright 380 mm, high 200 mm; cages from with running wheels 270 mm) and all rats in the same room (olfactory socialization was possible), Harlan Teklad Global 18% Protein Rodent diet was used, and the rats were fed ad libidum. Rats were transferred into cages with running wheels and one rat per cage during the (active) night period. Running wheels were connected to a bike computer that calculated the number of rotations and time in use which could be translated into km by quantification of the circumference of the wheels. Rats were purchased from Harlan Laboratories Bioservice GmbH; Walrode, Germany.

You wrote: 5. Rat genes are conventionally written in italics, and only the first letter is capitalized (i.e., Sod, Cat...) instead of SOD, CAT, etc. Please correct the gene abbreviations in the text and images.

Response: We agree and have corrected these gene names.

You wrote: At the end of the text, delete the sections Appendix A and B.

Response: We have corrected the abbreviation list and removed the non-used Appendices.

 

 

Reviewer 2 Report

Comments and Suggestions for Authors

The authors worked with female Wistar (WIS; n=6 - control group) and female SHR rats. The female SHRs were divided into two groups: sedentary SHR-S (n=21) group and SHR-R group (n=15), which had a voluntary access to running wheels during their active night-time. By the way, it remains unclear to me why the authors write that the experiment was performed on 46 rats?After all, 6 + 15 + 21 = 42. Then, in connection with the choice of females for the experiment, I would like to ask what the authors had in mind when they wrote “….ethical reasons motivated us to restrict our analysis to female rats” (line #295).

At the beginning of the experiment the rats were 6 weeks old, at the end – 7.5 months old. After ending the experiment, the rats were euthanized and three brain, regions - the cortex, medulla oblongata, and olfactory bulbs were chosen for genetic analyses. The RT-PCR was performed to study the expression of 25 genes each playing their own role in the following processes: oxidative stress (SOD2, CAT), glucose transporting processes (SLC2A1, SLC2A4), inflammation (CCL2, IL6), blood brain perfusion (AGTR1, AGTR2, ACE1, 319 ACE2, REN, END1, ENDRB, ECE1, NPPA), uncoupling proteins (UCP2, UCP3, SLC25A27, 320 SLC25A14), and dementia (PSEN1, PSEN2, SG2, APP, BACE1, NSE). Most genes that correlated with blood pressure were detected in the olfactory bulb (10 genes), brain cortex (7 genes) and in medulla oblongata (5 genes). However, only the expression of UCP2 gene showed approximately the same score of negative correlation with blood pressure levels in all three brain regions. UCP2 gene encodes a mitochondrial membrane protein that dissipates the proton gradient to reduce reactive oxygen species (ROS) and regulate metabolism. Reduction of UCP2 gene expression in hypertensive rats may indicate on persistent oxidative stress. Data of Western Blots of the UCP2 protein expression confirms this result mainly for medulla and cortex. However, enhanced physical activity in SHR-R rats did not increase the mRNA expression of UCP2 and, in accordance with this, to reduce supposed oxidative stress. The authors themselves confirm the results obtained with the UCP2 gene as “… a main new finding of our study is that hypertension downregulates the expression of UCP2 in various areas of the brain” (line #208). Interestingly, the expression of genes CAT and REN in the medulla was increased in hypertensive rats, although the increase of physical activity have no any influence on it. The running activity modified cortical genes expression such as UCP3, which was up regulated (positive correlation with running), and ACE1, VEGFA, APP, NSE was downregulated (negative correlation with running, by the way, the negative correlations must be marked with minus, see the legend to the Fig. 6 – lines #166, 167 ). Then, I stumbled over the RAG gene; the authors did not study it in this article (?) (line#153).

The presence of oxidative stress in the cerebral cortex of hypertensive rats may be confirmed by more intensive DHE-staining of cortical slices. Moreover, this indicator decreases in hypertensive rats with increased physical activity.

As for the all genes studied by the authors, of course, many different interesting versions have been put forward about their involvement in possible disturbances of the brain function in the presence of high blood pressure, which is accompanied by decreased or increase of the levels of physical activity. But the whole problem is that these genes presented as if they are isolated from each other, like “peas in a bag” without any functionally bonds between them. In this case, it would be appropriate to use principal component analysis and the constructions of the genetic networks to analyze the gene interactions and to identify the key metabolomic processes, which make the main contribution to connections of the high blood pressure with the levels of physical activity in determination of development of senile dementia and/or Alzheimer's disease.

 

 

Author Response

We thank the reviewer for critical evaluation of our manuscript and study. Please find enclosed a point-by-point response to your suggestions.

You wrote: The authors worked with female Wistar (WIS; n=6 - control group) and female SHR rats. The female SHRs were divided into two groups: sedentary SHR-S (n=21) group and SHR-R group (n=15), which had a voluntary access to running wheels during their active night-time. By the way, it remains unclear to me why the authors write that the experiment was performed on 46 rats?After all, 6 + 15 + 21 = 42. Then, in connection with the choice of females for the experiment, I would like to ask what the authors had in mind when they wrote “….ethical reasons motivated us to restrict our analysis to female rats” (line #295).

Response: Sorry for the confusion in some of the figure legends and the abstract. We have corrected the total number of rats. In summary, 10 (not 6) normotensive Wistar rats were included in this study (therefore 46 rats) as stated correctly in the M&M part (part 4). Please note that in agreement with the comment of your co-reviewer we have now carefully checked all animal and samples number used in this study.

Female ats were used exclusively in this study for scientific reasons as explained in detail in the introduction. In addition, we were asked by the ethical board the must approve the study to restrict to one sex as otherwise we would have a doubling in animal numbers whereas the scientific question is restricted to female rats. Therefore, male rats were excluded from this study for both scientific and ethical reasons. Consequently, we could not address the additional question whether the observed differences between male and female sex in clinical studies (see introduction) is mirrored by this model. To avoid any confusion, we deleted this summary sentence.

You wrote: At the beginning of the experiment the rats were 6 weeks old, at the end – 7.5 months old. After ending the experiment, the rats were euthanized and three brain, regions - the cortex, medulla oblongata, and olfactory bulbs were chosen for genetic analyses. The RT-PCR was performed to study the expression of 25 genes each playing their own role in the following processes: oxidative stress (SOD2, CAT), glucose transporting processes (SLC2A1, SLC2A4), inflammation (CCL2, IL6), blood brain perfusion (AGTR1, AGTR2, ACE1, 319 ACE2, REN, END1, ENDRB, ECE1, NPPA), uncoupling proteins (UCP2, UCP3, SLC25A27, 320 SLC25A14), and dementia (PSEN1, PSEN2, SG2, APP, BACE1, NSE). Most genes that correlated with blood pressure were detected in the olfactory bulb (10 genes), brain cortex (7 genes) and in medulla oblongata (5 genes). However, only the expression of UCP2 gene showed approximately the same score of negative correlation with blood pressure levels in all three brain regions. UCP2 gene encodes a mitochondrial membrane protein that dissipates the proton gradient to reduce reactive oxygen species (ROS) and regulate metabolism. Reduction of UCP2 gene expression in hypertensive rats may indicate on persistent oxidative stress. Data of Western Blots of the UCP2 protein expression confirms this result mainly for medulla and cortex. However, enhanced physical activity in SHR-R rats did not increase the mRNA expression of UCP2 and, in accordance with this, to reduce supposed oxidative stress. The authors themselves confirm the results obtained with the UCP2 gene as “… a main new finding of our study is that hypertension downregulates the expression of UCP2 in various areas of the brain” (line #208). Interestingly, the expression of genes CAT and REN in the medulla was increased in hypertensive rats, although the increase of physical activity have no any influence on it. The running activity modified cortical genes expression such as UCP3, which was up regulated (positive correlation with running), and ACE1, VEGFA, APP, NSE was downregulated (negative correlation with running, by the way, the negative correlations must be marked with minus, see the legend to the Fig. 6 – lines #166, 167 ). Then, I stumbled over the RAG gene; the authors did not study it in this article (?) (line#153).

Response: RAG gene was missing in the listing of the Alzheimer’s disease genes. This is now added. Thanks for finding this missing listing.

You wrote: The presence of oxidative stress in the cerebral cortex of hypertensive rats may be confirmed by more intensive DHE-staining of cortical slices. Moreover, this indicator decreases in hypertensive rats with increased physical activity.

Response: We agree to your point that running activity reduces oxidative stress in SHRs.

You wrote: As for the all genes studied by the authors, of course, many different interesting versions have been put forward about their involvement in possible disturbances of the brain function in the presence of high blood pressure, which is accompanied by decreased or increase of the levels of physical activity. But the whole problem is that these genes presented as if they are isolated from each other, like “peas in a bag” without any functionally bonds between them. In this case, it would be appropriate to use principal component analysis and the constructions of the genetic networks to analyze the gene interactions and to identify the key metabolomic processes, which make the main contribution to connections of the high blood pressure with the levels of physical activity in determination of development of senile dementia and/or Alzheimer's disease.

Response: Construction of genetic networks is mostly used in screening studies to identify relationships in an unbiased way. However, this study used the opposite approach: We focused exclusively on genes that have already been discussed to play a role in the pathophysiological process. We agree that the genes and their products do not work independently. As such, we identified genes involved in three pathways: Oxidative stress defense (Ucp3, Ace1), vascularization (Vegfa), and association to Alzheimer’s disease (App, Nse; all Fig. 6). Therefore, although the gens that’s expression was quantified here based on PCR analysis may look as peas in a bag as are nevertheless grouped to specific pathways and well interesting targets. Overall, we expect that the upregulation of Ucp3 and the downregulation of Ace1 contribute to the reduction of oxidative stress (seen in Fig. 8), that the downregulation of HIF-dependent gene Vegfa indicates as better vascularization (together with ET1 expression), and that the downregulation of App and Nse suggested a reduced risk for the development of Alzheimer’s disease.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have corrected all points in the text of MS