Serum and Striatal Redox and Metabolic Responses to Progesterone Treatment in Rats with Common Carotid Ligation

Round 1

Reviewer 1 Report

The manuscript investigates the effects of progesterone (P4) on systemic and central redox and metabolic alterations in a rat model of chronic cerebral hypoperfusion (CCH). The study addresses a relevant and timely topic, given the increasing interest in neuroprotective strategies targeting oxidative stress and metabolic dysfunction in cerebrovascular and neurodegenerative disorders.

The authors present a comprehensive set of biochemical analyses in both serum and striatal compartments, along with functional assessment of sensorimotor performance. The integrative approach linking systemic and local responses represents a strength of the study.

However, while the manuscript is well-structured and the experimental design is generally sound, several important issues related to mechanistic interpretation, methodological clarity, and data interpretation should be addressed before the manuscript can be considered for publication.

1. The manuscript proposes that progesterone exerts its effects through modulation of purinergic signaling (CD39/CD73), mitochondrial metabolism, and phospholipase A2 (PLA2) activity. However, these mechanisms are not directly assessed in the study. The involvement of CD39/CD73 is inferred from nucleotide hydrolysis data, but no direct measurements (e.g., enzyme expression or activity assays) are provided. Similarly, claims regarding mitochondrial function and PLA2 regulation remain speculative. The authors should either (i) provide experimental evidence supporting these pathways or (ii) clearly state these interpretations as hypotheses rather than conclusions.
2. The study relies on a single time point (7 days post-treatment), which restricts the understanding of the temporal dynamics of progesterone’s effects. It remains unclear whether the observed changes represent early adaptive responses, peak effects, or partial recovery. The limitation of using a single time point should be more explicitly acknowledged and discussed in terms of its impact on data interpretation.
3. The increase in SOD activity in the striatum is interpreted as a protective effect of progesterone. However, elevated antioxidant enzyme activity may also reflect a compensatory response to persistent oxidative stress rather than recovery. Alternative interpretations should be considered and discussed.
4. The study relies exclusively on biochemical and functional data. No histological or immunohistochemical analyses are provided to confirm structural preservation or neuronal protection. This limitation should be more clearly emphasized, and its implications discussed.
5. Although improvements in sensorimotor performance are reported, the magnitude of these changes appears modest. It is unclear whether these improvements are biologically meaningful or only statistically significant. The authors should better contextualize the functional relevance of these findings.
6. The exclusive use of male rats should be acknowledged as a limitation, particularly given the hormonal nature of the intervention.
7. Why did the authors not assess the activity of glutathione peroxidase (GPx)? Considering that GPx plays a central role in cellular antioxidant defense by reducing hydrogen peroxide and lipid hydroperoxides—particularly under conditions of moderate oxidative stress—it is essential for complementing the actions of superoxide dismutase (SOD) and catalase (CAT). Moreover, the manuscript itself suggests a potential involvement of GPx in H₂O₂ detoxification at the synaptic level, yet no direct measurement was performed. The lack of GPx activity assessment represents a significant limitation, as it prevents a more comprehensive understanding of the enzymatic antioxidant response and the mechanisms underlying the reported neuroprotective effects.
8. The authors should include, in the Methods section, the date of approval by the ethics committee.

In summary, the manuscript provides valuable insights into the systemic and central effects of progesterone in a model of chronic cerebral hypoperfusion. However, the conclusions would benefit from a more cautious interpretation of mechanistic pathways, improved discussion of limitations, and clarification of methodological aspects.

1. The authors should include, in the Methods section, the date of approval by the ethics committee.

Author Response

The manuscript investigates the effects of progesterone (P4) on systemic and central redox and metabolic alterations in a rat model of chronic cerebral hypoperfusion (CCH). The study addresses a relevant and timely topic, given the increasing interest in neuroprotective strategies targeting oxidative stress and metabolic dysfunction in cerebrovascular and neurodegenerative disorders. The authors present a comprehensive set of biochemical analyses in both serum and striatal compartments, along with functional assessment of sensorimotor performance. The integrative approach linking systemic and local responses represents a strength of the study. However, while the manuscript is well-structured and the experimental design is generally sound, several important issues related to mechanistic interpretation, methodological clarity, and data interpretation should be addressed before the manuscript can be considered for publication.

Comment 1: The manuscript proposes that progesterone exerts its effects through modulation of purinergic signaling (CD39/CD73), mitochondrial metabolism, and phospholipase A2 (PLA2) activity. However, these mechanisms are not directly assessed in the study. The involvement of CD39/CD73 is inferred from nucleotide hydrolysis data, but no direct measurements (e.g., enzyme expression or activity assays) are provided. Similarly, claims regarding mitochondrial function and PLA2 regulation remain speculative. The authors should either (i) provide experimental evidence supporting these pathways or (ii) clearly state these interpretations as hypotheses rather than conclusions.

Response 1: We thank the Reviewer for this important and insightful comment. We agree that the proposed involvement of purinergic signaling (activities and/or expression of CD39/CD73), mitochondrial metabolism, and PLA2 activity is not directly demonstrated in the present study. These mechanisms were inferred based on the observed changes in nucleotide and lipid metabolism and previously published data. Accordingly, we have revised the manuscript to clearly present these pathways as hypothetical mechanisms rather than definitive conclusions. The text has been modified throughout the section Discussion to avoid overstatements as interpretations are now explicitly framed as potential mechanisms rather than causal conclusions, and this limitation has been explicitly acknowledged. Changes in the revised version of the manuscript are marked in red.

Comment 2: The study relies on a single time point (7 days post-treatment), which restricts the understanding of the temporal dynamics of progesterone’s effects. It remains unclear whether the observed changes represent early adaptive responses, peak effects, or partial recovery. The limitation of using a single time point should be more explicitly acknowledged and discussed in terms of its impact on data interpretation.

Response 2: We agree that the use of a single time point represents a limitation of the study, as it does not allow assessment of the temporal dynamics of progesterone (P4) effects. This time point was selected based on our previous studies indicating that it corresponds to a peak of metabolic and neuropharmacological alterations in this model in other vulnerable brain regions (hippocampus and prefrontal cortex). We acknowledge that the observed changes may reflect a specific phase of the response, and this limitation has now been explicitly pointed out in the revised version of the manuscript and marked in red.

Stanojlović, M.; Guševac, I.; Grković, I.; Mitrović, N.; Horvat, A.; Drakulić, D. Time-related sex differences in cerebral hypoperfusion-induced brain injury. Arch. Biol. Sci. 2014, 66, 1673–1680.

Stanojlović, M.; Horvat, A.; Guševac, I.; Grković, I.; Mitrović, N.; Buzadžić, I.; Drakulić, D. Time course of cerebral hypoperfusion-induced neurodegenerative changes in the cortex of male and female rats. Folia Biol. (Praha) 2014, 60, 123–132.

Stanojlović, M.; Guševac Stojanović, I.; Zarić, M.; Martinović, J.; Mitrović, N.; Grković, I.; Drakulić, D. Progesterone Protects Prefrontal Cortex in Rat Model of Permanent Bilateral Common Carotid Occlusion via Progesterone Receptors and Akt/Erk/eNOS. Cell. Mol. Neurobiol. 2020, 40, 829–843.

Stanojlović, M.; Guševac, I.; Grković, I.; Zlatković, J.; Mitrović, N.; Zarić, M.; Horvat, A.; Drakulić, D. Effects of chronic cerebral hypoperfusion and low-dose progesterone treatment on apoptotic processes, expression and subcellular localization of key elements within Akt and Erk signaling pathways in rat hippocampus. Neuroscience 2015, 311, 308–321.

 

Comment 3: The increase in SOD activity in the striatum is interpreted as a protective effect of progesterone. However, elevated antioxidant enzyme activity may also reflect a compensatory response to persistent oxidative stress rather than recovery. Alternative interpretations should be considered and discussed.

Response 3: We agree with the Reviewer’s important remark that the increased SOD activity may reflect not only an enhanced antioxidant defense and protective effect but also a compensatory response to persistent oxidative stress. The section Discussion has been revised to include this alternative interpretation and adjusted accordingly to avoid the overstatement.

 

Comment 4: The study relies exclusively on biochemical and functional data. No histological or immunohistochemical analyses are provided to confirm structural preservation or neuronal protection. This limitation should be more clearly emphasized, and its implications discussed.

Response 4: In accordance with the Reviewer’s suggestion, we have addressed this limitation in the revised version of the manuscript. Specifically, we have explained how our sample size was ethically guided by the 3Rs principles to achieve sufficient statistical power for biochemical and functional analyses, thereby limiting the tissue available for morphological studies. We have also revised the section Discussion to clarify that our current findings should be interpreted as evidence of metabolic and redox preservation, which require further morphological validation to definitively establish structural neuroprotection.

 

Comment 5: Although improvements in sensorimotor performance are reported, the magnitude of these changes appears modest. It is unclear whether these improvements are biologically meaningful or only statistically significant. The authors should better contextualize the functional relevance of these findings.

Response 5: We thank the Reviewer for this thoughtful comment. Although the observed sensorimotor improvements were modest, they were highly consistent and statistically significant, likely reflecting early-stage functional recovery rather than full restoration, as reported in similar neurodegenerative models. Thus, we agree that the biological relevance of these changes should be interpreted with caution, and the section Discussion has been revised to better contextualize the functional findings in relation to the biochemical data. The changes in the revised version of the manuscript are marked in red.

 

Comment 6: The exclusive use of male rats should be acknowledged as a limitation, particularly given the hormonal nature of the intervention.

Response 6: We thank the Reviewer for this important observation. We acknowledge that the use of only male rats represents a limitation, particularly considering the hormonal nature of P4. This point has now been more explicitly stated in the revised version of the manuscript, while future studies should address potential sex-related differences.

 

Comment 7: Why did the authors not assess the activity of glutathione peroxidase (GPx)? Considering that GPx plays a central role in cellular antioxidant defense by reducing hydrogen peroxide and lipid hydroperoxides—particularly under conditions of moderate oxidative stress—it is essential for complementing the actions of superoxide dismutase (SOD) and catalase (CAT). Moreover, the manuscript itself suggests a potential involvement of GPx in H₂O₂ detoxification at the synaptic level, yet no direct measurement was performed. The lack of GPx activity assessment represents a significant limitation, as it prevents a more comprehensive understanding of the enzymatic antioxidant response and the mechanisms underlying the reported neuroprotective effects.

Response 7: We thank the Reviewer for this important and well-justified comment. We agree that glutathione peroxidase (GPx) plays a critical role in antioxidant defense, particularly in the detoxification of H2O2 and lipid hydroperoxides. The absence of GPx activity assessment represents a limitation of the present study, primarily related to technical and resource limitations at the time of the experimental phase. However, we prioritized the measurement of superoxide dismutase (SOD) and catalase (CAT) as the first line of enzymatic defense to establish the fundamental antioxidant profile in our model. In accordance with the Reviewer’s suggestion, we have revised the section Discussion to explicitly acknowledge the absence of GPx data as a limitation. We have also removed any speculative statements regarding GPx involvement to ensure that our conclusions remain strictly supported by the presented data.

 

Comment 8: The authors should include, in the Methods section, the date of approval by the ethics committee.

Response 8: The protocol numbers and date of Ethics Committee approvals have now been included in the sections Materials and Methods and Institutional Review Board Statement, according to the Reviewers’ concern.

 

Comment 9: In summary, the manuscript provides valuable insights into the systemic and central effects of progesterone in a model of chronic cerebral hypoperfusion. However, the conclusions would benefit from a more cautious interpretation of mechanistic pathways, improved discussion of limitations, and clarification of methodological aspects.

Response 9: We thank the Reviewer for the constructive comments. We believe that all concerns have been appropriately addressed and that these revisions have improved the clarity and interpretation of the manuscript.

 

Detailed comments: The authors should include, in the Methods section, the date of approval by the ethics committee.

Response: We appreciate the Reviewer for pointing out this omission. The protocol numbers and date of the Ethics Committee approvals have now been added to the section Materials and Methods of the revised version of the manuscript, as well as in the section Institutional Review Board Statement. The changes are marked in red.

Reviewer 2 Report

Reviewer’s report:

Title: Serum and Striatal Redox and Metabolic Responses to Progesterone Treatment in Rats with Common Carotid Ligation.

Comments and Suggestions:

The manuscript by Stojanović et al., investigated the protective effects of progesterone in rats with chronic cerebral hypoperfusion caused by carotid artery ligation. They found that progesterone treatment restored redox balance and metabolic homeostasis in both serum (by increased adenine nucleotide) and striatum (by promoting nucleotide catabolism). They concluded that progesterone has the potential to permanently ligate the bilateral common carotid arteries.

The manuscript is very well accomplished with good English grammar and sentence structure, but there are few points/suggestions which need to be addressed.

1. The authors have done the vehicle or P4 treatments which lasted for 7 consecutive days. Do they also tried to find the effect till 14 or 21- or 28-days post treatment?
2. Since the authors used progesterone dose of 1.7 mg/kg/day dissolved in commercial flax oil, does they also tested other doses which could have changed effect on the results? On what basis they used this dose?
3. Line 180: please mention the ration of chloroform/methanol/water for lipid extraction.
4. Figure 1: The axis labels are missing. please mention the y-axis labels of Total score of sensorimotor function.

 

This manuscript fulfills the requirements for publication in Antioxidants.

Reviewer’s report:

Title: Serum and Striatal Redox and Metabolic Responses to Progesterone Treatment in Rats with Common Carotid Ligation.

Comments and Suggestions:

The manuscript by Stojanović et al., investigated the protective effects of progesterone in rats with chronic cerebral hypoperfusion caused by carotid artery ligation. They found that progesterone treatment restored redox balance and metabolic homeostasis in both serum (by increased adenine nucleotide) and striatum (by promoting nucleotide catabolism). They concluded that progesterone has the potential to permanently ligate the bilateral common carotid arteries.

The manuscript is very well accomplished with good English grammar and sentence structure, but there are few points/suggestions which need to be addressed.

1. The authors have done the vehicle or P4 treatments which lasted for 7 consecutive days. Do they also tried to find the effect till 14 or 21- or 28-days post treatment?
2. Since the authors used progesterone dose of 1.7 mg/kg/day dissolved in commercial flax oil, does they also tested other doses which could have changed effect on the results? On what basis they used this dose?
3. Line 180: please mention the ration of chloroform/methanol/water for lipid extraction.
4. Figure 1: The axis labels are missing. please mention the y-axis labels of Total score of sensorimotor function.

 

This manuscript fulfills the requirements for publication in Antioxidants.

Author Response

Comment 1: The manuscript by Stojanović et al., investigated the protective effects of progesterone in rats with chronic cerebral hypoperfusion caused by carotid artery ligation. They found that progesterone treatment restored redox balance and metabolic homeostasis in both serum (by increased adenine nucleotide) and striatum (by promoting nucleotide catabolism). They concluded that progesterone has the potential to permanently ligate the bilateral common carotid arteries. The manuscript is very well accomplished with good English grammar and sentence structure, but there are few points/suggestions which need to be addressed.

Response 1: We appreciate the comment. We would like to emphasize that progesterone (P4) was not used to induce carotid artery ligation, but rather as a therapeutic agent administered following permanent bilateral carotid occlusion (CCH).

 

Comment 2: The authors have done the vehicle or P4 treatments which lasted for 7 consecutive days. Do they also tried to find the effect till 14 or 21- or 28-days post treatment?

Response 2: We thank the Reviewer for raising this important question. In the current study, we have focused on a single time point (7 days post-surgery and treatment), which was selected based on our previous studies demonstrating that this interval corresponds to a peak of metabolic and neuropharmacological alterations in this experimental model in other brain regions (e.g., hippocampus and prefrontal cortex). This design allowed us, currently, to focus on a well-defined phase of maximal metabolic disturbance, which we in parallel specifically examined in the striatum and serum. Although we agree that inclusion of additional time points would provide a more extensive understanding of the progression and persistence of the observed effects, this was beyond the scope of the present study. Our future studies will be designed to address the long-term effects of P4 treatment.

Stanojlović, M.; Guševac, I.; Grković, I.; Mitrović, N.; Horvat, A.; Drakulić, D. Time-related sex differences in cerebral hypoperfusion-induced brain injury. Arch. Biol. Sci. 2014, 66, 1673–1680.

Stanojlović, M.; Horvat, A.; Guševac, I.; Grković, I.; Mitrović, N.; Buzadžić, I.; Drakulić, D. Time course of cerebral hypoperfusion-induced neurodegenerative changes in the cortex of male and female rats. Folia Biol. (Praha) 2014, 60, 123–132.

Stanojlović, M.; Guševac Stojanović, I.; Zarić, M.; Martinović, J.; Mitrović, N.; Grković, I.; Drakulić, D. Progesterone Protects Prefrontal Cortex in Rat Model of Permanent Bilateral Common Carotid Occlusion via Progesterone Receptors and Akt/Erk/eNOS. Cell. Mol. Neurobiol. 2020, 40, 829–843.

Stanojlović, M.; Guševac, I.; Grković, I.; Zlatković, J.; Mitrović, N.; Zarić, M.; Horvat, A.; Drakulić, D. Effects of chronic cerebral hypoperfusion and low-dose progesterone treatment on apoptotic processes, expression and subcellular localization of key elements within Akt and Erk signaling pathways in rat hippocampus. Neuroscience 2015, 311, 308–321.

 

Comment 3: Since the authors used progesterone dose of 1.7 mg/kg/day dissolved in commercial flax oil, does they also tested other doses which could have changed effect on the results? On what basis they used this dose?

Response 3: We thank the Reviewer for the opportunity to clarify the rationale for dose selection. We selected the dose of P4 (1.7 mg/kg/day) based on our previous studies, in which this regimen demonstrated neuroprotective effects in different brain regions (e.g., hippocampus and prefrontal cortex). In the present study, we aimed to further investigate the effects of this previously validated neuroprotective dose, rather than to conduct a dose–response analysis. We agree that evaluation of different doses as well as post‒treatment days could provide additional insight and represent an important direction for our future research.

Stanojlović, M.; Guševac Stojanović, I.; Zarić, M.; Martinović, J.; Mitrović, N.; Grković, I.; Drakulić, D. Progesterone Protects Prefrontal Cortex in Rat Model of Permanent Bilateral Common Carotid Occlusion via Progesterone Receptors and Akt/Erk/eNOS. Cell. Mol. Neurobiol. 2020, 40, 829–843.

Stanojlović, M.; Guševac, I.; Grković, I.; Zlatković, J.; Mitrović, N.; Zarić, M.; Horvat, A.; Drakulić, D. Effects of chronic cerebral hypoperfusion and low-dose progesterone treatment on apoptotic processes, expression and subcellular localization of key elements within Akt and Erk signaling pathways in rat hippocampus. Neuroscience 2015, 311, 308–321.

 

Comment 4: Line 180: please mention the ration of chloroform/methanol/water for lipid extraction.

Response 4: We appreciate the comment. The lipid extraction procedure has been inserted in the revised version of the manuscript by specifying the solvent ratio as chloroform/methanol/water (2:1:0.4, v/v/v), which ensures proper phase separation and efficient recovery of lipids in the organic phase, in accordance with the Folch method. The change in the revised version of the manuscript is marked in red.

 

Comment 5: Figure 1: The axis labels are missing. please mention the y-axis labels of Total score of sensorimotor function.

Response 5: We thank the Reviewer for this helpful comment. As suggested by Reviewer 2# and Reviewer 3#, to improve clarity and readability, Figure 1. has been replaced with Table 1., which presents the data in a more accessible and transparent format.

 

Reviewer 3 Report

The main question addressed by the work is to study the effects of progesterone on the attenuation of ischemic stroke consequences in an animal model.

The topic is a subject of intense current research of stroke therapy. It is an important field that is required to fill the gap in our medical ability to mitigate severe consequences of brain vessel blockade.

The study adds to our understanding of progesterone’s role in both cell antioxidant defense and adenine metabolism with a particular focus on striatum. The weak point of the study is that there is no comparison of striatum response to that of other region(s) of the brain affected by carotid artery ligation.

PG4 was injected s/c for slow systemic delivery. The model used is supposed to cause injury of all brain regions. Please explain the choice of brain region striatum investigated rather than full brain tissue or several regions for comparison. It’s not clear.

• Method 2.3: indicate the route of vehicle/PG4 injection.
• Lanes 150-151, 165-166: cite the original publications that describe details of the protocols with any additional changes made.
• Figure 1. Indicate the units on Score scale, indicate number of animals (is n=6 or less?). The figure is difficult to read. The readers would be mostly interested to see the change between 0 and 7 days for every group.
• Lane 294: ‘promote… and promotion of pro-inflammatory signaling’ should be read ‘promote… and pro-inflammatory signaling’
• Lane 331-333: Tissue catalase activity does not necessarily reflect hydrogen peroxide level in the tissue. Thus, the conclusion on antioxidant enzyme Cat and GPx roles here is not warranted by the data present.

Additional comments: In the Methods section, in many cases authors cite their previous work that used the same techniques in place of the original references with detailed description of the methods. Thus, to find the method for PAB used in the manuscript, a reader would have to open two intermediate self-citations before finding the original paper. This issue should be fixed.

Author Response

Major comment: The main question addressed by the work is to study the effects of progesterone on the attenuation of ischemic stroke consequences in an animal model. The topic is a subject of intense current research of stroke therapy. It is an important field that is required to fill the gap in our medical ability to mitigate severe consequences of brain vessel blockade. The study adds to our understanding of progesterone’s role in both cell antioxidant defense and adenine metabolism with a particular focus on striatum. The weak point of the study is that there is no comparison of striatum response to that of other region(s) of the brain affected by carotid artery ligation. PG4 was injected s/c for slow systemic delivery. The model used is supposed to cause injury of all brain regions. Please explain the choice of brain region striatum investigated rather than full brain tissue or several regions for comparison. It’s not clear.

Major response: We sincerely thank the Reviewer for raising this important issue. Analyzing whole-brain tissue would likely dilute and mask localized biochemical shifts, as the specific "signal" from affected areas could be obscured by less affected tissue. Therefore, we focused exclusively on the striatum, which is especially vulnerable to CCH due to its high metabolic demand and specific vascular supply. Moreover, it should be noted that the preparation of the crude synaptosomal fraction, a unique biological compartment, is both time- and technically demanding. To ensure a sufficient yield for a broad set of redox, lipid, and adenine metabolism analyses from a single animal, the study was restricted to this specific anatomical region. Processing multiple regions separately would have introduced major technical constraints during fraction isolation, prolonged sample handling times, and potentially compromised the biochemical integrity and consistency of the preparation. Finally, while a comparative analysis within multiple brain regions would certainly be of interest and could provide additional insight into region-specific effects, this was not the primary focus of the present study and represents an important direction for future research.

Liu, Y.J.; Chen, J.; Li, X.; Zhou, X.; Hu, Y.M.; Chu, S.F.; Peng, Y.; Chen, N.H. Research progress on adenosine in central nervous system diseases. CNS Neurosci. Ther. 2019, 25, 899–910.

Stanojlović, M.; Guševac Stojanović, I.; Zarić, M.; Martinović, J.; Mitrović, N.; Grković, I.; Drakulić, D. Progesterone Protects Prefrontal Cortex in Rat Model of Permanent Bilateral Common Carotid Occlusion via Progesterone Receptors and Akt/Erk/eNOS. Cell. Mol. Neurobiol. 2020, 40, 829–843.

Stanojlović, M.; Guševac, I.; Grković, I.; Zlatković, J.; Mitrović, N.; Zarić, M.; Horvat, A.; Drakulić, D. Effects of chronic cerebral hypoperfusion and low-dose progesterone treatment on apoptotic processes, expression and subcellular localization of key elements within Akt and Erk signaling pathways in rat hippocampus. Neuroscience 2015, 311, 308–321.

 

Comment 1: Method 2.3: indicate the route of vehicle/PG4 injection.

Response 1: We thank the Reviewer for pointing this out. Both vehicle and P4 were administered subcutaneously once daily for 7 consecutive days. The route of administration has now been explicitly stated in the revised version of the manuscript, in the section Materials and Methods, while change is marked in red.

 

Comment 2: Lanes 150-151, 165-166: cite the original publications that describe details of the protocols with any additional changes made.

Response 2: We appreciate this suggestion. The original publications describing, in short, the applied protocols for oxidative stress parameters have now been added in the revised version of the manuscript. Additionally, a relevant reference has been added to the description in lines 165–166 to further clarify the methodology. Both sections are paraphrased and changes in revised version of the manuscript are marked in red.

 

Comment 3: Figure 1. Indicate the units on Score scale, indicate number of animals (is n=6 or less?). The figure is difficult to read. The readers would be mostly interested to see the change between 0 and 7 days for every group.

Response 3: We thank the Reviewer for this helpful comment. To improve clarity and readability, Figure 1. has been replaced with Table 1., which presents the data in a more accessible and transparent format. The number of animals per group (n = 6) is now explicitly indicated in both Figures legends as well in Table 1. Legend, while the scoring system has been clarified by specifying that the total neurological score ranges from 0 to 10 (arbitrary units) in the Table 1. Legend, as described in the section Materials and Methods. Consequently, the numbering of all subsequent figures has been adjusted accordingly. All changes in the revised manuscript are marked in red.

To address the Reviewer's interest and demonstrate the progression of the sensorimotor deficit, the complete dataset for all assessed time points (days 0, 1, 3, and 7) is provided below (Table R1.). The time points were selected according to the literature data describing the temporal dynamics of this experimental model. Pre-operative neurological assessment was conducted to confirm that all experimental animals exhibited normal sensorimotor function and that no pre-existing differences existed between individuals prior to surgical procedures and treatments. Obtained data show that animals in the 2VO+V group exhibited persistent neurological impairment, while P4 treatment following 2VO led to a gradual improvement over time, with partial recovery observed by day 7.

Table R1. Total neurological scores (arbitrary units) on days 0, 1, 3, and 7.

Group	Day 0	Day 1	Day 3	Day 7
Sham+V	10.00±0.00	8.50±0.22	8.83±0.40	9.17±0.31
2VO+V	10.00±0.00	5.00±0.26∗∗∗	5.17±0.31∗∗∗	5.83±0.31∗∗∗
2VO+P4	10.00±0.00	5.83±0.40∗∗∗	6.83±0.48∗∗#	7.83±0.31∗###

While additional time points were recorded to monitor progression, the biochemical analyses were specifically designed for the 7-day endpoint. To maintain consistency between functional and biochemical datasets, intermediate time points are provided herein for transparency but were not included in the main manuscript.

Stanojlović, M.; Guševac, I.; Grković, I.; Mitrović, N.; Horvat, A.; Drakulić, D. Time-related sex differences in cerebral hypoperfusion-induced brain injury. Arch. Biol. Sci. 2014, 66, 1673–1680.

Stanojlović, M.; Horvat, A.; Guševac, I.; Grković, I.; Mitrović, N.; Buzadžić, I.; Drakulić, D. Time course of cerebral hypoperfusion-induced neurodegenerative changes in the cortex of male and female rats. Folia Biol. (Praha) 2014, 60, 123–132.

Farkas, E., Luiten, P. G., & Bari, F. (2007). Permanent, bilateral common carotid artery occlusion in the rat: a model for chronic cerebral hypoperfusion-related neurodegenerative diseases. Brain Res Rev. 2007, 54(1), 162–180.

 

Comment 4: Lane 294: ‘promote… and promotion of pro-inflammatory signaling’ should be read ‘promote… and pro-inflammatory signaling’.

Response 4: The part of this paragraph is changed according to Reviewer 1# and Reviewer 3# suggestion. The change is marked in red in the revised version of the manuscript.

 

Comment 5: Lane 331-333: Tissue catalase activity does not necessarily reflect hydrogen peroxide level in the tissue. Thus, the conclusion on antioxidant enzyme Cat and GPx roles here is not warranted by the data present.

Response 5: We thank the Reviewer for this critical and insightful comment. We agree that catalase (CAT) activity alone does not directly reflect hydrogen peroxide (H202) levels in the tissue, while conclusions regarding the relative contribution of CAT and GPx should be interpreted with caution in the absence of direct measurements of H2O2 or GPx activity. Accordingly, we have revised the manuscript to avoid the overstatement. The section Discussion has been modified to clearly state that the proposed involvement of GPx in H2O2 detoxification is speculative and should be confirmed by future studies. The changes in the revised version of the manuscript are marked in red.

 

Additional comment: In the Methods section, in many cases authors cite their previous work that used the same techniques in place of the original references with detailed description of the methods. Thus, to find the method for PAB used in the manuscript, a reader would have to open two intermediate self-citations before finding the original paper. This issue should be fixed.

Additional response: We thank the Reviewer for this valid point. We apologize for the inconvenience caused by the use of intermediate self-citations. In accordance with the suggestion, we have revised the section Materials and Methods to include the original references not only for the PAB assay, but also for AOPP, and LPO assays, along with a more detailed description of the procedures. All other methodological citations have also been reviewed to ensure that original references are prioritized. All changes are marked in red in the revised version of the manuscript.

Round 2

Reviewer 1 Report

The authors addressed the suggestions and satisfactorily answered all the concerns raised, with the requested revisions to the text and results.

The authors addressed the suggestions and satisfactorily answered all the concerns raised, with the requested revisions to the text and results.

Reviewer 3 Report

All critical points were addressed. Thank you for nice study.

none