Chronic Inhibition of Nitric Oxide Synthases Impairs Spatiotemporal Learning and Memory to a Similar Extent in C57BL/6 and hAPP23+/− Mice

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

1) The authors did not clearly mention how much was the time of treatments. They stated a 16-week treatment in line 71, while they stated in lines 72-75 that the measurements were done in first and second week and after that animals were killed. This must be corrected and clearly explained. It must be also stated why there was a relatively large time gap (one week) between the final assessments.

2) Why L-NAME was administered to the animals via drinking water, instead of injection. Injection could be more precise and reduce variation among the groups and individual animals. However, administration via drinking water is less aggressive. How the authors overcome the variations including time of drinking and the amount of drinking?

3) It seems that the authors have done no baseline measurement in MWM, Blood pressure, Echocardiography and PWV tests. This was necessary for a precise and reliable comparison between the groups.

4) Words such as LVIDM LVPW, IVRT or any other abbreviation within the manuscript must be written in the first mention, while the abbreviation is placed in parentheses. Then, the abbreviations can be used alone in next mentions.

5) In captions of figures, it is not common to use p<0.0001 and it should be mentioned as p<0.001.

6) In line 256-258 the authors stated: “A decreased trend could be observed in the ven- 256 tral cerebral cortex for L-NAME treated hAPP223+/- mice compared to untreated 257 hAPP223+/- mice, which the authors attribute to the large distribution of data points.”

7) It was better to do further complementary studies in this regard. Especially considering that L-NAME made this effect in both hippocampus and cortex and both C57BL/6 and hAPP23+/-.

8) The authors did not assess the levels of NO or at least “total antioxidant capacity” in the studied brain areas. This could help to interpret the data more efficiently. It was also better to assess the expression level of nitric oxide synthase in transcription, translation, and functional levels.

Comments on the Quality of English Language

The English language quality is suitable. 

Author Response

The authors would like to mention that the document has undergone significant rewording and reduction in length. This was done to address the high resemblance to our previous manuscripts and adhere to word count restrictions.

 REVIEWER 1: Comments and Suggestions for Authors

1) The authors did not clearly mention how much was the time of treatments. They stated a 16-week treatment in line 71, while they stated in lines 72-75 that the measurements were done in first and second week and after that animals were killed. This must be corrected and clearly explained. It must be also stated why there was a relatively large time gap (one week) between the final assessments.

The authors better indicated the timings of the experiment as follows on Lines 75 – 81:

A two-week experimental series followed 15 weeks of L-NAME treatment. In the first week, during the 15th week of treatment, spatial learning and memory were assessed using the Morris water maze (MWM) test. In the second week, corresponding to the 16th week of treatment, blood pressure and echocardiographic analyses were per-formed. At week 16, the mice were euthanized under deep anesthesia with sodium pentobarbital (250 mg/kg, i.p. [21]) to allow ex vivo assessment of arterial stiffness (AS) and histological analysis of cerebral amyloidosis.

2) Why L-NAME was administered to the animals via drinking water, instead of injection. Injection could be more precise and reduce variation among the groups and individual animals. However, administration via drinking water is less aggressive. How the authors overcome the variations including time of drinking and the amount of drinking?

The authors wish to emphasize that intraperitoneal injections were not the preferred method of administration, as they are considered an aggressive approach for pharmacological treatment, and they are suboptimal for long-term treatments. We would like to direct your attention to our previous work (Hendrickx, Jhana O., et al. "Serum corticosterone and insulin resistance as early biomarkers in the hAPP23 overexpressing mouse model of Alzheimer’s disease." International journal of molecular sciences (2021)), where we discussed the chronically increased cortisol levels in hAPP23+/- mice. Since injections can be a relatively aggressive method, this could potentially have a negative impact on the already stressed AD mouse model, potentially affecting not only cardiovascular but also neurological outcomes.

Furthermore, it's important to acknowledge that in every study, certain types of variations can be challenging to control. These variations are reflected in the data points where variability is naturally expected in each animal group.

3) It seems that the authors have done no baseline measurement in MWM, Blood pressure, Echocardiography and PWV tests. This was necessary for a precise and reliable comparison between the groups.

The authors want to emphasize that, for each animal group subjected to pharmacological treatment, corresponding control groups were included. This resulted in a total of four animal groups being studied. Furthermore, it's important to note that both C57BL/6 and hAPP23 animals have been extensively examined in previous research, providing valuable reference data.

Hendrickx, Jhana O., et al. "Altered stress hormone levels affect in vivo vascular function in the hAPP23+/− overexpressing mouse model of Alzheimer’s disease." American Journal of Physiology-Heart and Circulatory Physiology 321.5 (2021): H905-H919.

De Moudt, Sofie, et al. "Progressive aortic stiffness in aging C57Bl/6 mice displays altered contractile behaviour and extracellular matrix changes." Communications Biology 5.1 (2022): 605.

Hendrickx, Jhana O., et al. "Age-related cognitive decline in spatial learning and memory of C57BL/6J mice." Behavioural brain research 418 (2022): 113649.

4) Words such as LVIDM LVPW, IVRT or any other abbreviation within the manuscript must be written in the first mention, while the abbreviation is placed in parentheses. Then, the abbreviations can be used alone in next mentions.

The authors would like to clarify that the full version of the manuscript was not properly uploaded to the platform. The reviewed manuscript was lacking Table 1, and we would like to direct your attention to the table's legend on lines 215 - 224, which contains all the necessary abbreviations.

5) In captions of figures, it is not common to use p<0.0001 and it should be mentioned as p<0.001.

Changes were made accordingly throughout the manuscript.

6) In line 256-258 the authors stated: “A decreased trend could be observed in the ven- 256 tral cerebral cortex for L-NAME treated hAPP223+/- mice compared to untreated 257 hAPP223+/- mice, which the authors attribute to the large distribution of data points.”

The authors refer to the answer on comment 7, below regarding this comment.

7) It was better to do further complementary studies in this regard. Especially considering that L-NAME made this effect in both hippocampus and cortex and both C57BL/6 and hAPP23+/-.

We've incorporated the subsequent section into the discussion, acknowledging it as a limitation of this study and emphasizing the necessity for additional research on this subject.

Lines 383 – 388: However, we did not detect a significant increase in amyloid load in the brains of hAPP23+/- mice following L-NAME treatment. It is important to note that data variability was notable in both hippocampal and cortical tissue samples of hAPP23+/- mice, likely due to the relatively small sample size. We acknowledge this as a limitation and stress the need for more comprehensive studies on cerebral amyloidosis resulting from L-NAME treatment.

8) The authors did not assess the levels of NO or at least “total antioxidant capacity” in the studied brain areas. This could help to interpret the data more efficiently. It was also better to assess the expression level of nitric oxide synthase in transcription, translation, and functional levels.

The authors acknowledge the above comment as a limitation of this study and incorporated the following section in the discussion on lines 319- 323:

However, we did not conduct an in-depth analysis of NO levels or the transcriptional and translational NOS expression in the specific brain regions studied. Therefore, we suggest conducting more comprehensive studies to thoroughly assess NO levels, NOS expressions, and total antioxidant levels for a better understanding of the subject.

Reviewer 2 Report

Comments and Suggestions for Authors

In the current study authors brought to light a potential mechanistic connection between cardiovascular disease and neurodegeneration. This connection places specific emphasis on the relationship between arterial stiffness (AS) and Alzheimer's disease (AD). Consequently, authors embarked on a study aimed at understanding the consequences of prolonged inhibition of nitric oxide synthase (NOS) isoforms, a factor contributing to AS, on neurobehavioral performance. Additionally, authors sought to discern whether these effects differed in an AD model compared to control mice.

 

In pursuit of this objective, both C57BL/6 and hAPP23+/- mice (a widely recognized AD model) were administered a solution containing 0.5 mg/mL N(G)-Nitro-L-Arginine Methyl Ester (L-NAME) in their drinking water for a period of 16 weeks. Their research findings indicate that the chronic, non-selective inhibition of NOS resulted in a significant increase in AS, along with a decline in spatio-temporal learning and memory capabilities in both C57BL/6 and hAPP23+/- mice. Notably, these effects were consistent in both groups, underscoring the role of neuronal NOS (nNOS) in cognitive aging, irrespective of a genetic predisposition to AD.

 

Study is good and I recommend for publication after some minor changes.

1.     Sex of the animals is not disclosed, if they belong to same sex, is there any reason to select?

2.     In the line 70, average L-NAME intake is given, please provide the SD value.

3.     In line 146, please use subscript to write CO₂ and O₂.

4.     In Figure 3, please add the track diagrams generated by the tracking software.

 

5.     In Figure 4 A and B, please discuss why hAPP23^+/- treated animals have large variability than the control animals.

Author Response

REVIEWER 2: Comments and Suggestions for Authors

Study is good and I recommend for publication after some minor changes.

1. Sex of the animals is not disclosed, if they belong to same sex, is there any reason to select?

The authors indicated the sex of animals on Line 61. We preferred the use of male mice in our experiment, in part due to concern that the hormone cycle in females would cause behavioral variation that could negatively impact the behavioral assessment performed in this study.

2. In the line 70, average L-NAME intake is given, please provide the SD value.

The average L-NAME intake ± SEM was added on Lines 73-74 accordingly.

3. In line 146, please use subscript to write CO₂and O₂.

The requested change was made accordingly on Line 145 of the manuscript.

4. In Figure 3, please add the track diagrams generated by the tracking software.

We regret to inform reviewer 2 that, due to the considerable time that has elapsed since the experiment and the expiration of data storage, we are unable to provide the track diagrams for this behavioral assessment.

5. In Figure 4 A and B, please discuss why hAPP23^+/-treated animals have large variability than the control animals.

The authors acknowledge this observation and direct Reviewer 2 to the newly added section in the discussion, located at lines 383 – 388, where this difference is more comprehensively explained, emphasizing the necessity for further research:

Lines 383 – 388: However, we did not detect a significant increase in amyloid load in the brains of hAPP23+/- mice following L-NAME treatment. It is important to note that data varia-bility was notable in both hippocampal and cortical tissue samples of hAPP23+/- mice, likely due to the relatively small sample size. We acknowledge this as a limitation and stress the need for more comprehensive studies on cerebral amyloidosis resulting from L-NAME treatment.

Reviewer 3 Report

Comments and Suggestions for Authors

An interesting and well executed investigation which once again shows how significantly the inhibition of NO synthase affects the state of the cardiovascular system, and also shows that non-selective inhibition of NOS activity with L-NAME induces not only an increase in arterial stiffness but also a decrease in hippocampal-dependent spatiotemporal learning and memory in mice. These results suggest that neuronal NOS may affect the cognitive aging independently on a genetic predisposition to Alzheimer’s disease.

Two problems.

First, I can’t understand why was the inhibition of NO synthase activity not accompanied by an increase in pressure? A number of studies carried out at the end of the last century and the beginning of this century showed that in all mammals (including humans) adding of L-NAME to the drinking water (and therefore the decrease in NO production by vascuilar endothelium and an increase in peripheral vascular resistance) induce hypertension. I think that this problem should be scholarly discussed.

Second. The Table 1 didn't load in my version of the manuscript (maybe computer problems). There is a fairly informative legend to it, but there is no table itself. This made it impossible to evaluate the results of echocardiographic analysis.

 

Author Response

REVIEWER 3: Comments and Suggestions for Authors

An interesting and well executed investigation which once again shows how significantly the inhibition of NO synthase affects the state of the cardiovascular system, and also shows that non-selective inhibition of NOS activity with L-NAME induces not only an increase in arterial stiffness but also a decrease in hippocampal-dependent spatiotemporal learning and memory in mice. These results suggest that neuronal NOS may affect the cognitive aging independently on a genetic predisposition to Alzheimer’s disease.

Two problems.

First, I can’t understand why was the inhibition of NO synthase activity not accompanied by an increase in pressure? A number of studies carried out at the end of the last century and the beginning of this century showed that in all mammals (including humans) adding of L-NAME to the drinking water (and therefore the decrease in NO production by vascuilar endothelium and an increase in peripheral vascular resistance) induce hypertension. I think that this problem should be scholarly discussed.

First and foremost, the authors would like to emphasize that the y-axes in Figure 1 A and B were intentionally chosen with a wide range to enhance a back to back visualization of systolic and diastolic blood pressure data for all the animals under study.

Secondly, we would like to refer to our prior work ( De Moudt et. al., 2022. Aortic stiffness in L-NAME treated C57Bl/6 mice displays a shift from early endothelial dysfunction to late-term vascular smooth muscle cell dysfunction. Frontiers in Physiology) where a similar experimental procedure involving a 16-week L-NAME treatment in C57BL/6 mice was conducted. In that study, we demonstrated that arterial stiffness preceded the development of peripheral hypertension, which progressively increased after 4 weeks of treatment, leading to elevated systolic and diastolic blood pressure. These findings are in alignment with our current results, where aPWV data also precede the onset of hypertension.

Thirdly, it is important to clarify that we used a lower dosage of L-NAME in comparison to other studies. This lower dosage was chosen to achieve only partial inhibition of eNOS activity in the aortic vessel wall. This intentional choice may have had an impact on the absence of a fully developed hypertensive phenotype.  

Second. The Table 1 didn't load in my version of the manuscript (maybe computer problems). There is a fairly informative legend to it, but there is no table itself. This made it impossible to evaluate the results of echocardiographic analysis.

The authors would like to clarify that the full version of the manuscript was not properly uploaded to the platform. The reviewed manuscript was lacking Table 1, and we would like to direct your attention to Table 1 starting on Line 215. 

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

1) As it was said in the previous comments, words such as LVIDM LVPW, IVRT or any other abbreviation within the manuscript must be written in the first mention, while the abbreviation is placed in parentheses. Then, the abbreviations can be used alone in next mentions. putting them in table 1 is good, but they also must be written as recommended above in the first mention within the manuscript. This is a rule in scientific writing to avoid misunderstanding and confusion of the readers.

2) In the new revision, the authors refrained from correcting most of the comments and limited themselves to providing explanations in this field, although their explanations were often insufficient and unacceptable. Especially the comments No. 7 and 8, which demanded more attention and were considered as serious flaws of this study, but the authors limited themselves to saying confessional statements about the existence of these flaws in the discussion section and did not try to fix them. However, considering the overall value of the work and the acceptable structure of the draft, and the fact that the correction of the mentioned comments could greatly contribute to the richness of the work, but it requires the re-doing of extensive laboratory work, I consider the manuscript in its current version to be printable. In addition, in my opinion, the study has the minimum qualities necessary for publication, and the failure to fix the mentioned comments does not make it ineligible.

Author Response

1) As it was said in the previous comments, words such as LVIDM LVPW, IVRT or any other abbreviation within the manuscript must be written in the first mention, while the abbreviation is placed in parentheses. Then, the abbreviations can be used alone in next mentions. putting them in table 1 is good, but they also must be written as recommended above in the first mention within the manuscript. This is a rule in scientific writing to avoid misunderstanding and confusion of the readers.

The authors specified the echocardiographic abbreviations on lines 208-209 'increased left ventricular inner diameters (LVID) and left ventricular posterior wall thicknesses (LVPW) and decreased ejected left ventricular (LV) volumes' and line 214 'isovolumetric retention time (IVRT) ' of the manuscript. 

2) In the new revision, the authors refrained from correcting most of the comments and limited themselves to providing explanations in this field, although their explanations were often insufficient and unacceptable. Especially the comments No. 7 and 8, which demanded more attention and were considered as serious flaws of this study, but the authors limited themselves to saying confessional statements about the existence of these flaws in the discussion section and did not try to fix them. However, considering the overall value of the work and the acceptable structure of the draft, and the fact that the correction of the mentioned comments could greatly contribute to the richness of the work, but it requires the re-doing of extensive laboratory work, I consider the manuscript in its current version to be printable. In addition, in my opinion, the study has the minimum qualities necessary for publication, and the failure to fix the mentioned comments does not make it ineligible.

We express our gratitude to Reviewer 1 for his/her sincerity and thoughtful evaluation in support of the publication.