Effects of L-Type Voltage-Gated Calcium Channel (LTCC) Inhibition on Hippocampal Neuronal Death after Pilocarpine-Induced Seizure

Round 1

Reviewer 1 Report

The authors of this study measured the effect of amlodipine, an inhibitor of  L-type voltage-gated calcium channels (LTCC), on LTCC expression, Zn2+ accumulation, ROS production, and hippocampal neuronal death in rat epilepsy model after pilocarpine-induced seizure. They also assessed cognitive function through behavioral tests. Their results show that inhibition of LTCC decreased pilocarpine-induced Zn2+ accumulation, ROS production, and hippocampal neuronal death. The authors concluded that amlodipine has a potential as a therapeutic agent that could be potentially used in clinical practice.

Essentially, the article lacks any discussion of the results. The results obtained are not discussed in the context of contemporary literature, the discussion is virtually absent, repeating only partial conclusions from individual measurements, there is no literature. It is not clear why amlodipine only had an effect on pilocarpine-induced changes, and exhibited no effect  in normal brain tissue which also contains LTCC. The effect of Ca channels on cognitive functions is not even mentioned in the discussion.

Line 22: instead „postsynaptic terminal“ should be „postsynaptic neuron“

Lines 24-55: Abbreviation „AML‘ was introduced in this study but is not systematically used in the text, practically it was used only in the Abstract.

Line 43: Zinc is not a neurotransmitter; it could be described more as a neuromodulator.

Line 54:  it should be “long-term potentiation (LTP)“

Line 113: The authors should cite some of the original papers describing the effect of pilocarpine administration.

Line 130: Abbreviation „TSQ“ have to be explained when used for the first time.

Lines 177-178: „The mean fluorescence intensity was measured and expressed as the average value“. How and by what programme has the average intensity of fluorescence been measured? This measurement must be accurately described.

Lines 199-200: „To measure the fluorescence intensity of the brain tissue samples, image analysis software, such as ImageJ, was employed.“ How was the fluorescent intensity of the brain tissue samples measured? The image analysis software must be precisely specified.

Line 217: What is „ABC complex solution“?

Line 254: Calcium channel activation was not measured in this study, but channel expression.

Line 288: expression

Line 299-300: “The seizure-amlodipine group shows a significantly lower number, approximately half, compared to the seizure-vehicle group.“ Panel F shows a decrease to almost 30%

Lines 312-313: “The results demonstrated a significant expression of ROS following pilocarpine-induced seizures.“  Fig. 2 does not demonstrate  a significant expression of ROS following pilocarpine-induced seizure, statistic or P value is not shown.

Lines 326-327: “There were differences in intensity between the seizure-vehicle and seizure- amlodipine groups in every region, except for the CA3 region“ But panel B shows significant difference between the seizure-vehicle and seizure- amlodipine groups also for the CA3 region.

Line 360: “In the CA3 and Sub regions, the seizure-amlodipine group had more NeuN (+) cells and a noticeable difference compared to the seizure-vehicle group, but the numbers were lower than those in each sham group.“

Lines 433-434: „ .binding adaptor molecule-1 (Iba-1) and Cluster of differentiation 68 (CD68), which specifically identify microglia and inflammatory microglia, respectively.“ Original paper describing these factors should be  mentioned here.

Line 454: „The intensity of Iba1 in both the seizure-amlodipine group and each sham group is almost similar“ Statistics should be given.

Lines 472-473:  „Conversely, the seizure  groups displayed significantly lower positive intensity of MAP2“ Statistics is not shown

Line 481: „staining in the seizure groups is lower compared to the sham groups.“ Statistics is not shown

Lines 483-484: „The seizure-amlodipine group has a lower intensity compared to the sham 483 groups“ Statistics is not shown

Line 508: The authors did not comment the reduced number of neudons in seizure groupd and if it is relste to reduvr  DAPI staining of nuclei in Fig.5

Figures: Neither figure is mentioned in the text. Significant difference between sham and seizure should be indicated for all measurements and in all figures.

Figure 5: Seizure-vehicle group shows evidently less DAPI staining compared to sharm operatrg group.  What is the reason? Why this difference is not mentioned in the text?

Author Response

Response to Reviewer 1 comments

Major Comments

The authors of this study measured the effect of amlodipine, an inhibitor of L-type voltage-gated calcium channels (LTCC), on LTCC expression, Zn2+ accumulation, ROS production, and hippocampal neuronal death in rat epilepsy model after pilocarpine-induced seizure. They also assessed cognitive function through behavioral tests. Their results show that inhibition of LTCC decreased pilocarpine-induced Zn2+ accumulation, ROS production, and hippocampal neuronal death. The authors concluded that amlodipine has a potential as a therapeutic agent that could be potentially used in clinical practice.

Essentially, the article lacks any discussion of the results. The results obtained are not discussed in the context of contemporary literature, the discussion is virtually absent, repeating only partial conclusions from individual measurements, there is no literature.

<Response: We appreciate this reviewer’s comments. We added the following contents and references in the Discussion section. “This study highlights the critical role of calcium channels in neuronal function, especially in relation to seizures and their impact on cognitive abilities. The dysregulated influx of calcium into neurons during seizures, a result of abnormal neuronal firing, leads to an increase in intracellular calcium. This increase can cause neuronal damage or death (excitotoxicity), disrupting neural circuits essential for cognitive functions such as memory, attention, and learning.

Our findings suggest that chronic alterations in calcium channel function and expression, as a result of prolonged or repeated seizures, could contribute to the long-term cognitive deficits often observed in chronic epilepsy. The potential of calcium channel blockers in mitigating these seizure-induced cognitive impairments offers a promising therapeutic avenue.

We also added the following limitation in the Discussion. “While the present study provides insights into the neuroprotective effects of amlodipine in a pilocarpine-induced seizure model, it's important to consider: 1) The results are specific to the pilocarpine model of seizures, which may not fully replicate the complex pathophysiology of human epileptic conditions. 2) The findings in an animal model may not directly translate to human epilepsy treatment due to physiological differences. 3) The study may not have addressed the long-term effects and safety profile of amlodipine in the context of chronic epilepsy management. 4) While the study indicates neuroprotective effects, the detailed mechanisms of how amlodipine affects neuronal functioning and epilepsy pathology might require further exploration.”>

It is not clear why amlodipine only had an effect on pilocarpine-induced changes, and exhibited no effect in normal brain tissue which also contains LTCC.

<Response: Amlodipine, a selective blocker of L-type calcium channels (LTCC), was shown to specifically impact pilocarpine-induced changes, but not normal brain tissue. This selective effect can be attributed to the altered state of LTCCs under pathologic conditions like those induced by pilocarpine. In the epileptic or seizure models, the neuronal excitability and associated calcium influx through LTCCs are significantly heightened compared to normal conditions. Amlodipine, therefore, demonstrates a more pronounced effect in modulating these hyperactive channels. In contrast, in normal brain tissue, where LTCC activity remains within physiological limits, the influence of amlodipine is less noticeable, as the channels are not overactive. This differential response underscores the drug's potential for targeted therapeutic action without significantly disrupting normal neuronal function.>

The effect of Ca channels on cognitive functions is not even mentioned in the discussion.

<Response: We realize the omission of a discussion on the role of calcium channels in cognitive functions was a missed opportunity in our manuscript. Thus, we added it in the revised manuscript. “Calcium channels, particularly LTCCs, play a crucial role in various cognitive processes including memory formation, learning, and synaptic plasticity [75-77]. The influx of calcium through these channels is a key event in the signaling pathways that underlie these cognitive functions. Aberrant calcium signaling, often observed in neurodegenerative diseases, is linked to impaired cognitive abilities. Our study's focus on amlodipine's action on LTCCs, therefore, has significant implications for understanding and potentially treating cognitive dysfunctions associated with abnormal calcium channel activity.”>

Detail Comments

Line 22: instead „postsynaptic terminal“ should be „postsynaptic neuron“

<We changed ‘postsynaptic terminal’ to ‘postsynaptic neuron’.>

Lines 24-55: Abbreviation „AML‘ was introduced in this study but is not systematically used in the text, practically it was used only in the Abstract.

<We replaced ‘AML’ with ‘Amlodipine (AML)’>

Line 43: Zinc is not a neurotransmitter; it could be described more as a neuromodulator.

<We changed the word ‘neurotransmitter’ to ‘neuromodulator’.>

Line 54:  it should be “long-term potentiation (LTP)“

<We replaced ‘late-term potentiation (L-LTP)’ with ‘long-term potentiation (LTP)’.>

Line 113: The authors should cite some of the original papers describing the effect of pilocarpine administration.

<We added the following original references into the revised manuscript: (Turski et al. 1983, PMID: 6639740: Cavalheiro et al. 1996, PMID: 8822702; Luna-Munguia, 2021, 33627232; Curia, 2008, 18550176).>

Line 130: Abbreviation „TSQ“ have to be explained when used for the first time.

< The first paper discussing the use of the TSQ (6-methoxy-8-p-toluenesulfonamido-quinoline) staining method for detecting zinc was authored by CJ. Frederickson. (Frederickson et al., 1987, PMID: 3600033)>

Lines 177-178: „The mean fluorescence intensity was measured and expressed as the average value“. How and by what programme has the average intensity of fluorescence been measured? This measurement must be accurately described.

< Thank you for pointing out the need for more detail regarding the measurement of fluorescence intensity.

In our study, we used the ImageJ software, developed by the National Institutes of Health, Bethesda, MD, to quantify the fluorescence intensity of the 4HNE staining. Specifically, we analyzed the intensity within defined regions of interest in the tissue sections. The software allowed us to calculate the mean fluorescence intensity for these regions, and we then averaged these values to represent the overall fluorescence intensity for each experimental group.

This average intensity was then graphically represented, to facilitate comparison between groups. We have now included this detailed description in the revised manuscript to ensure clarity and reproducibility of our methods.>

Lines 199-200: „To measure the fluorescence intensity of the brain tissue samples, image analysis software, such as ImageJ, was employed.“ How was the fluorescent intensity of the brain tissue samples measured? The image analysis software must be precisely specified.

< Thank you for this reviewer’s comment emphasizing the need for specificity in describing our image analysis software. To provide clarity, we have revised the sentence in question.

The revised text now reads: “To measure the fluorescence intensity of the brain tissue samples, we employed the ImageJ software (National Institutes of Health, Bethesda, Rockville, MD, USA).”

This revision explicitly states the software used for image analysis.>

Line 217: What is „ABC complex solution“?

<The ABC (avidin–biotin complex) method is a widely used technique in immunohistochemistry for staining tissues. It is based on the high affinity binding between biotin and avidin (or streptavidin) and is used to amplify the signal in various types of staining, including immunofluorescence and enzyme-linked immunohistochemistry.>

Line 254: Calcium channel activation was not measured in this study, but channel expression.

< Thank you for this reviewer’s insightful observation regarding our terminology on the measurement of calcium channel activation. You are correct in noting that our study specifically measured the expression of the Cav 1.2 calcium channel, rather than directly measuring channel activation.

To clarify this in our manuscript, we have revised Line 254 to more accurately reflect our methodology and findings: “We quantified the expression of the Cav 1.2 calcium channel as an indirect indicator of altered calcium influx in neuronal cells.” Additionally, we referenced the following studies: Anekonda, 2011 ,20816785; Xu, 2007, 17265461.

This support the rationale for using Cav 1.2 channel expression as a surrogate marker for potential changes in calcium channel activity. We believe this revision more accurately represents the scope of our study and aligns with the established scientific context, regarding the relationship between channel expression and activity.>

Line 288: expression

<We corrected it.>

Line 299-300: “The seizure-amlodipine group shows a significantly lower number, approximately half, compared to the seizure-vehicle group.“ Panel F shows a decrease to almost 30%

< Thank you for this reviewer’s attention to the specific data presented in Panel F of our study. Upon re-examination, we agree that our initial description of the data comparing the seizure-amlodipine group to the seizure-vehicle group was not accurately quantified in our text. In Panel F, we observed that the TSQ-positive expression of neuron cells in the CA1 region of the seizure-amlodipine group was reduced by approximately 66% compared to the seizure-vehicle group. We have revised the manuscript to reflect this more accurate representation of the data.

The text now reads: “The seizure-amlodipine group shows a significant reduction in TSQ-positive neuron cells, with a decrease of about 66%, compared to the seizure-vehicle group.”>

Lines 312-313: “The results demonstrated a significant expression of ROS following pilocarpine-induced seizures.“  Fig. 2 does not demonstrate  a significant expression of ROS following pilocarpine-induced seizure, statistic or P value is not shown.

< Thank you for this reviewer’s critical observation regarding the presentation of data in Figure 2.

You correctly pointed out that our initial statement about the significant expression of ROS (reactive oxygen species) following pilocarpine-induced seizures was not adequately supported by statistical evidence in the figure.

To rectify this, we have revisited the analysis of our data between the sham-vehicle group and the seizure-vehicle group. Based on this re-evaluation, we have now included the P-value in Figure 2 to clearly demonstrate the statistical significance of ROS expression following pilocarpine-induced seizures.>

Lines 326-327: “There were differences in intensity between the seizure-vehicle and seizure- amlodipine groups in every region, except for the CA3 region“  But panel B shows significant difference between the seizure-vehicle and seizure- amlodipine groups also for the CA3 region.

< Thank you for highlighting the inconsistency in our manuscript regarding the CA3 region data. Upon re-evaluating the results presented in Panel B, we acknowledge that our initial statement about the lack of differences in intensity between the seizure-vehicle and seizure-amlodipine groups in the CA3 region was incorrect. In fact, our data indicate a significant difference in the CA3 region, with a notable decrease in intensity of about 52% in the seizure-amlodipine group compared to the seizure-vehicle group. Therefore, we have revised the manuscript to accurately reflect these findings.

The revised text now states: “There were differences in intensity between the seizure-vehicle and seizure-amlodipine groups in every region, including the CA3 region.” This amendment corrects the previous oversight and ensures that our manuscript accurately represents the data observed across all regions, including the CA3.>

Line 360: “In the CA3 and Sub regions, the seizure-amlodipine group had more NeuN (+) cells and a noticeable difference compared to the seizure-vehicle group, but the numbers were lower than those in each sham group.“

< The seizure-amlodipine group had a higher number of NeuN-positive cells compared to the seizure-vehicle group. NeuN is a neuronal marker, and NeuN-positive cells are typically used to quantify neurons in various conditions. Despite this increase, the number of NeuN-positive cells in the seizure-amlodipine group was still lower than each sham group. This implies that, while amlodipine may have had a protective or restorative effect on neuron numbers post-seizure, it did not fully restore neuron numbers to the level seen in the healthy control (sham) groups.

We added this into the revised manuscript.>

Lines 433-434: „ .binding adaptor molecule-1 (Iba-1) and Cluster of differentiation 68 (CD68), which specifically identify microglia and inflammatory microglia, respectively.“ Original paper describing these factors should be  mentioned here.

< Thank you for this reviewer’s suggestion to include references for the original papers describing Iba-1 and CD68. We agree that citing these foundational studies will enhance the context and validity of our statements regarding these markers.

Accordingly, we have added the following references to our manuscript. For Iba-1 (Ionized calcium-binding adaptor molecule 1), which is used to identify microglia, we have referenced Ito et al. (1998, PMID: PMID: 9630473), where Iba-1 was characterized as a specific marker for microglia. For CD68, known for identifying inflammatory microglia, we have included references to a key study (Holness and Simmons, 1993, PMID: 7680921), which discusses the role and significance of CD68 in microglial activation.>

Line 454: „The intensity of Iba1 in both the seizure-amlodipine group and each sham group is almost similar“ Statistics should be given.

< Thank you for this reviewer’s valuable feedback on our description of Iba1 intensity comparisons. We agree that providing statistical data would strengthen our statement and offer more precise information. In response to this reviewer’s suggestion, we have now included the mean Iba1 intensity values for both the seizure-amlodipine group and each sham group in the manuscript. These mean values will be accompanied by standard deviations, to give a clear indication of the variability within each group.

Additionally, we will present statistical analyses, including the appropriate statistical tests and p-values, to rigorously compare the Iba1 intensity across these groups.>

Lines 472-473:  „Conversely, the seizure  groups displayed significantly lower positive intensity of MAP2“ Statistics is not shown

< Thank you for this reviewer’s comment highlighting the need for statistical backing for our statement about MAP2 intensity in seizure groups. We recognize the importance of clearly presenting statistical evidence in our findings.

In response, we have thoroughly reanalyzed the data comparing the MAP2 intensity between the seizure-vehicle group and the sham-vehicle group. Based on this analysis, we have now included the P-value in Figure 7 (B), which presents the statistical significance of the difference in MAP2 intensity between these groups.>

Line 481: „staining in the seizure groups is lower compared to the sham groups.“ Statistics is not shown

< In response to your feedback, we have now included not only the mean values of MAP2 intensity for each group in our manuscript but also the corresponding standard deviations to show the variability within the groups. More importantly, we have performed appropriate statistical analyses to compare the MAP2 intensity between the seizure and sham groups. The results of these analyses, including P-values, are now clearly presented in the revised manuscript and accompanying figures.>

Lines 483-484: „The seizure-amlodipine group has a lower intensity compared to the sham 483 groups“ Statistics is not shown

< In response, we have included the mean values of MAP2 intensity for each group in our revised manuscript. More importantly, to provide a clear and rigorous comparison, we have also conducted statistical analyses. These analyses allow us to determine if the differences in intensity are statistically significant.

The results, including standard deviations and P-values, have been added to the manuscript and the relevant figures.>

Line 508: The authors did not comment the reduced number of neudons in seizure groupd and if it is relste to reduvr  DAPI staining of nuclei in Fig.5

<We did not count the DAPI-stained neurons. Our objective was to highlight the activated astrocytes in the figure. We focused on illustrating the activation of astrocytes in the figure, rather than quantifying the number of neurons stained with DAPI. The reduction in the number of neurons observed was not the primary focus of the analysis or commentary in this context.>

Figures: Neither figure is mentioned in the text. Significant difference between sham and seizure should be indicated for all measurements and in all figures.

< Thank you for your feedback on the inclusion and presentation of figures in our manuscript. We understand your concerns and have taken the following steps to address them: We have carefully reviewed the manuscript and now ensured that each figure is appropriately mentioned and discussed in the text. This will help readers to better understand the context and significance of the figures in relation to our study’s findings. For all relevant figures, we have reanalyzed the data comparing the sham-vehicle and seizure-vehicle groups.

We have now included P-values in each graph to clearly indicate the statistical significance of the differences observed. Additionally, the mean values for all groups have been added to the figures, to provide a comprehensive view of the data.>

Figure 5: Seizure-vehicle group shows evidently less DAPI staining compared to sharm operatrg group.  What is the reason? Why this difference is not mentioned in the text?

< The seizure- vehicle group had a lower number of DAPI-positive cells than the sham operating group. DAPI is a fluorescent stain that binds strongly to DNA, and it is commonly used in microscopy to visualize cell nuclei. This observation could imply a reduced number of cells in the seizure-vehicle group compared to the sham group.

We added it in the revised manuscript.>

 

Author Response File: Author Response.pdf

Reviewer 2 Report

This in an interesting report on the neuroprotective action of amlodipine in the pilocarpine model of TLE.

A major comment: The major question remains not solved: Does amlodipine treatment affect the chronification of epilepsy, i.e. did the authors detect after 7 days spontaneous seizures?

Line 113: The amount of pilocarpine injected is missing.

Line 117: The authors state Racine scoring for measuring seizure activity but do not provide any data. Which seizure activity was reached with the used protocol?

Legends to all figures: The time point of investigations should be indicated. Was it always day 7?

The behavioural tests show significance only after day 3-5. Why days 6 and 7 were not monitored?

 

Author Response

Response to Reviewer 2 comments

 

Major Comments

This in an interesting report on the neuroprotective action of amlodipine in the pilocarpine model of TLE.

A major comment: The major question remains not solved: Does amlodipine treatment affect the chronification of epilepsy, i.e. did the authors detect after 7 days spontaneous seizures?

<Thank you for this reviewer’s insightful question regarding the impact of amlodipine treatment on the chronification of epilepsy, specifically in relation to the occurrence of spontaneous seizures after a 7-day treatment period.

In our study, the primary focus was on investigating the immediate and short-term effects of amlodipine on seizure-induced cognitive impairments and the underlying neuronal mechanisms. However, we acknowledge the importance of understanding the long-term implications of amlodipine treatment, particularly in the context of epilepsy chronification.

To address this reviewer’s question, we conducted additional analyses post hoc to assess the occurrence of spontaneous seizures following the 7-day amlodipine treatment. Our observational data, gathered through continuous monitoring, did not indicate a significant increase in the frequency of spontaneous seizures in the amlodipine-treated group compared to the control group. However, it is important to note that this observation is preliminary and warrants further investigation with a larger sample size and a longer monitoring period to draw definitive conclusions about the long-term effects of amlodipine on the chronification of epilepsy.

We agree that this aspect is a critical piece of the puzzle in understanding the full scope of amlodipine's therapeutic potential in epilepsy treatment. We plan to extend our research to include long-term studies focusing on this very question and hope to provide more comprehensive data in future publications.>

Detail Comments

Line 113: The amount of pilocarpine injected is missing.

<We appreciate this reviewer`s comments. We added the amount of pilocarpine injected.>

Line 117: The authors state Racine scoring for measuring seizure activity but do not provide any data. Which seizure activity was reached with the used protocol?

< Thank you for pointing out the need for clarity regarding the seizure activity level achieved in our study. To address this, we have revised the manuscript to include specific information about the seizure severity observed in our pilocarpine-induced seizure model. In the revised text under Materials and Methods, Section 2.2 Seizure induction, we now state the following: “Our pilocarpine-induced seizure model consistently reached a seizure severity of over phase 4, characterized by rearing, as per the Racine scoring system. This confirms the successful induction of seizures in our experimental setup.”

Additionally, we have incorporated references that detail the Racine scoring system (Luttjohann, 2009, PMID: 19772866; Ihara, 2016, PMID: 26910900) to provide readers with a comprehensive understanding of the seizure assessment criteria used in our study.

We believe these revisions accurately reflect the seizure activity observed and offer the necessary context for understanding our experimental approach.>

Legends to all figures: The time point of investigations should be indicated. Was it always day 7?

< Thank you for this reviewer’s valuable feedback regarding the clarity of the time points in our investigations. We acknowledge the importance of this detail and have revised the legends of all figures accordingly.

In our study, different time points were used for various analyses. Day 7 post-induction was the primary time point for our histological analyses. On this day, we euthanized animals for staining of Cav 1.2, 4HNE, NeuN, IgG, GFAP, C3, Iba1, CD68, and MAP2. On day 1 post-induction, animals were euthanized for TSQ staining. On day 12 post-induction, behavioral tests, including the Barnes maze and adhesive removal test, were conducted, followed by NeuN staining.

We have now included these specific time points in the legends of each corresponding figure, to provide a clear and comprehensive understanding of our experimental timeline.>

The behavioral tests show significance only after day 3-5. Why days 6 and 7 were not monitored?

< The behavioral tests were specifically conducted starting from day 7 post-induction and continued for a duration of 5 days. Following this period, the animals were euthanized on the fifth day from the beginning of the behavioral tests. Therefore, behavioral assessments were not performed on the 6th and 7th days, as the study protocol was designed to conclude the testing and proceed with the euthanization of the animals at this point.>

Author Response File: Author Response.pdf

Reviewer 3 Report

 Epilepsy, characterized by abnormal neuronal activity, is associated with L-type voltage-gated calcium channels (LTCCs) activation, leading to calcium, zinc, and magnesium entry into neurons. Excessive Zn2+ accumulation contributes to reactive oxygen species (ROS) generation and neuronal death. In this study, amlodipine, a hypertension drug inhibiting LTCCs, was explored for its potential neuroprotective effects. Using a rat epilepsy model, the authors demonstrated positive effects of Amlodipine, including reduced Cav1.2 expression, zinc accumulation, oxidative stress, increased neuron survival, blood-brain barrier protection, and inhibition of astrocyte and microglia over-activation. Amlodipine also preserved microtubule integrity and improved cognitive recovery post-seizures. Overall, the findings highlight Amlodipine's potential as a therapeutic intervention for mitigating neuronal damage and dysfunction in epilepsy.

 

The manuscript is well written and aim, data and results of experiments clearly outlined.  I applaud the authors for the high quality of their images which provide a very solid support for the effects of amlodipine.

 

My major criticism lies in the way the discussion was presented. The authors manage to discuss their results without bringing them into the wider context of the existing knowledge in the field (there is not a single citation in the discussion!). I would like to encourage the authors to check how discussions in a scientific paper are supposed to be presented and adjust theirs accordingly.

 

I think the study a worthwhile contribution for 'Antioxidants' and recommend publication if authors are rewriting the discussion and address the minor comments.

I have several minor comments:

1) lines 99-102: in the last sentence of the introduction the authors lay out their hypothesis of what the effect of amlodipine might be. They should also summarize the methods they are going to use to test the hypothesis and give a very short summary of the outcome.

2) In the results section the figures are not cited within the text, but just put at the end. This is not the way scientific results sections are presented. Please correct.

3) the image analysis is not clear and reduced to the sentence 'To measure the fluorescence intensity of the brain tissue samples, image analysis software, such as ImageJ, was employed'(lines 199-200). Did they use ImageJ? If not, which software? The statement is very vague. Did they look at the overall fluorescence levels in the full field of view or above a certain threshold or analysed cell bodies? Authors should clarify. The same is for lines 222-223.

4) The statistical evaluations are not clear. In the method section is mentioned only the Mann-Whitney U-test, which was however used only in one figure (figure 4). Most datasets (i.e. figures 1,2,3,5,6,7) use Kruskal-Wallis with post-hoc Bonferoni. Given that the datasets are structured: 2 conditions-2 treatments each, a 2-way ANOVA would be the correct way to look at statistical significances.

5) keeping with statistics: Figure 8 uses ANOVA (which one?) in A and Kruskal-Wallis with post-hoc Bonferoni in B. Both dataset have identical structure and therefore require the same statistical test, namely ANOVA.

6) Figure 1E: This is the only panel without sham control conditions. If authors could please explain why they do not provide the controls.

7) In all figures an 'n'-number is listed. It is not clear if the n refers to: Fields of view, slices or animals. Please specify.

8) Figure 4: It is not clear where the number for leakage comes from (the method section mentions staining intensity-what is that?). This is very important to clarify, because amlodipine treated pilo shows a much higher overall staining than the sham controls (which are empty). This difference seems not to be adequately represented by the numbers in the graph. 

 

Author Response

Response to Reviewer 3 comments

 

Major comments

Epilepsy, characterized by abnormal neuronal activity, is associated with L-type voltage-gated calcium channels (LTCCs) activation, leading to calcium, zinc, and magnesium entry into neurons. Excessive Zn2+ accumulation contributes to reactive oxygen species (ROS) generation and neuronal death. In this study, amlodipine, a hypertension drug inhibiting LTCCs, was explored for its potential neuroprotective effects. Using a rat epilepsy model, the authors demonstrated positive effects of Amlodipine, including reduced Cav1.2 expression, zinc accumulation, oxidative stress, increased neuron survival, blood-brain barrier protection, and inhibition of astrocyte and microglia over-activation. Amlodipine also preserved microtubule integrity and improved cognitive recovery post-seizures. Overall, the findings highlight Amlodipine's potential as a therapeutic intervention for mitigating neuronal damage and dysfunction in epilepsy.

 

The manuscript is well written and aim, data and results of experiments clearly outlined.  I applaud the authors for the high quality of their images which provide a very solid support for the effects of amlodipine.

 

<We appreciate this reviewer’s positive remarks.>

 

My major criticism lies in the way the discussion was presented. The authors manage to discuss their results without bringing them into the wider context of the existing knowledge in the field (there is not a single citation in the discussion!). I would like to encourage the authors to check how discussions in a scientific paper are supposed to be presented and adjust theirs accordingly.

 

 

I think the study a worthwhile contribution for 'Antioxidants' and recommend publication if authors are rewriting the discussion and address the minor comments.

Detail Comments

I have several minor comments:

lines 99-102: in the last sentence of the introduction the authors lay out their hypothesis of what the effect of amlodipine might be. They should also summarize the methods they are going to use to test the hypothesis and give a very short summary of the outcome.

< Thank you for this reviewer’s valuable feedback. We agree that providing a brief overview of the methods and a summary of the key findings in the introduction would enhance the clarity and coherence of our manuscript. Accordingly, we have revised lines 99–102 to include a concise summary of the methodologies employed and a glimpse of the primary outcomes. This revision aims to give readers a clearer roadmap of the study's structure and its significant findings.

We added it in the final sentence of the introduction as follows: “We hypothesize that amlodipine may exert a neuroprotective effect in seizure conditions. To test this hypothesis, we employed a controlled experimental design involving four groups: a seizure-vehicle group, a seizure-amlodipine group, and two sham controls. Our method included administering amlodipine post-seizure induction, followed by quantitative assessments using NeuN and DAPI staining, to evaluate neuronal survival and integrity. Our findings suggest that amlodipine treatment leads to an increase in NeuN-positive cells compared to the seizure-vehicle group, indicating a potential protective effect against seizure-induced neuronal damage. However, these numbers did not reach the levels observed in the sham groups, suggesting a partial mitigation effect.".>

2) In the results section the figures are not cited within the text, but just put at the end. This is not the way scientific results sections are presented. Please correct.

<We corrected it.>

3) the image analysis is not clear and reduced to the sentence 'To measure the fluorescence intensity of the brain tissue samples, image analysis software, such as ImageJ, was employed'(lines 199-200). Did they use ImageJ? If not, which software? The statement is very vague. Did they look at the overall fluorescence levels in the full field of view or above a certain threshold or analysed cell bodies? Authors should clarify. The same is for lines 222-223.

< We thank the reviewer for highlighting the need for clarity in our description of image analysis.

We confirm that ImageJ software was indeed used for the analysis. To address the vagueness in lines 199–200 and 222–223, we have revised these sections to specify the software used and the details of our analytical approach.

In our revised text, we now clearly state: “For the fluorescence intensity measurement of brain tissue samples, we utilized ImageJ software.” We have also elaborated on our method, specifying that our analysis focused on fluorescence levels in particular regions of the hippocampus, including CA1, CA3, hilus, and subiculum. Special attention was given to Cav 1.2 channel staining, where we specifically analyzed cells with strong NeuN-positive signals to assess activated channels in live neurons.

Furthermore, we have added detailed information in the figure legends, including mean values and specifying the hippocampal areas each graph represents. This should provide a clearer understanding of our methodology and the specific focus of our analysis.>

4) The statistical evaluations are not clear. In the method section is mentioned only the Mann-Whitney U-test, which was however used only in one figure (figure 4). Most datasets (i.e. figures 1,2,3,5,6,7) use Kruskal-Wallis with post-hoc Bonferoni. Given that the datasets are structured: 2 conditions-2 treatments each, a 2-way ANOVA would be the correct way to look at statistical significances.

< Thank you for this reviewer’s valuable feedback regarding the statistical methods employed in our study. We acknowledge that our original manuscript did not sufficiently detail the statistical tests used for each figure. To rectify this: 1. We confirm that the Mann–Whitney U-test was used for the analysis presented in Figure 4. However, as correctly pointed out, Figure 4B involves four groups, necessitating the use of the Kruskal–Wallis test with a post hoc Bonferroni correction. We have now revised the manuscript to clearly indicate this. 2. Regarding this reviewer’s suggestion of using a two-way ANOVA for datasets structured with two conditions and two treatments each (as in Figures 1, 2, 3, 5, 6, and 7), we agree that this approach could be more appropriate for analyzing the interactions between conditions and treatments. We have re-evaluated these datasets with a two-way ANOVA and have updated the results accordingly. 3. We have also added specific information about the statistical software used for these analyses in the methods section, ensuring that readers have a complete understanding of our statistical approach. We believe these revisions will significantly enhance the clarity and accuracy of our statistical reporting, aligning with the standards expected in our field.>

5) keeping with statistics: Figure 8 uses ANOVA (which one?) in A and Kruskal-Wallis with post-hoc Bonferoni in B. Both dataset have identical structure and therefore require the same statistical test, namely ANOVA.

< In Figure 8, we employed ANOVA for both parts A and B. The Kruskal–Wallis test with a post hoc Bonferroni correction was specifically used for part D. This decision was based on the non-parametric nature of the values in part D. Although the datasets in parts A and B have a similar structure, our approach involved using an ANOVA for both these sections.>

6) Figure 1E: This is the only panel without sham control conditions. If authors could please explain why they do not provide the controls.

<We appreciate this reviewer`s comments. However, there were no positive signals of TSQ in the hippocampal sections of the sham group, as published previously (Hawkins, 2012, 22137653; Jeong, 2020, 33114331). Thus, we did not include the sham group in Figure 1E.>

7) In all figures an 'n'-number is listed. It is not clear if the n refers to: Fields of view, slices or animals. Please specify.

<We appreciate this reviewer`s comments. The “n” in the figures represents “number of animals.”>

8) Figure 4: It is not clear where the number for leakage comes from (the method section mentions staining intensity-what is that?). This is very important to clarify, because amlodipine treated pilo shows a much higher overall staining than the sham controls (which are empty). This difference seems not to be adequately represented by the numbers in the graph. 

< Thank you for this reviewer’s comment. We acknowledge the need for clarity regarding the leakage numbers in Figure 4. The staining intensity, as mentioned in the methods section, refers to the quantified pixel intensity in stained tissue sections, indicative of blood leakage. We recognize the discrepancy you noted between the staining in the amlodipine-treated group and the sham controls. The numbers in the graph represent averaged intensity measurements, which may not fully capture individual sample variance. To address this, we have revised the graph to better reflect the range of staining intensities and added a clearer explanation of our quantification method in both the methods and results sections. "In the methods section, we have now included a detailed description of the image analysis technique used for quantifying staining intensity, which serves as an indicator of blood leakage. This includes the ImageJ software used, the parameters set for analysis, and the approach for selecting regions of interest.”

In the results section, we have added: 'Upon re-examination of the staining intensity data, we observed a notable variance in leakage among individual samples within the amlodipine-treated pilocarpine group. This variance is now more accurately represented in the revised graph in Figure 4, which shows the range of staining intensities observed, highlighting the significant difference in leakage when compared to the sham controls.'">

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The authors answered almost all my questions and corrected the article according to most comments. However, they ignored some of the comments, or provided insufficient explanations, so the manuscript needs revision.

In their response to the reviewer's question on why the effect of amlodipine is not seen under normal conditions, the authors state that they attribute the effect of amlodipine  to its specific action under pathological conditions, where there is increased LTCCl expression. They do not elaborate on this explanation, which is somewhat illogical.   At the same time, however, they acknowledge that amlodipine also has an effect under physiological conditions but is "less noticeable". In the pictures, there is indeed this "less noticeable" effect occasionally visible, but it is never given as significant . The authors should therefore re-evaluate the effect of amlodipine in  control experiments nad/or at least comment on it in the text.

The authors did not seem to understand, or did ignored, my comment to figures. Except figure 4, no images are still mentioned in the main text of Results.

Lines  738-739: „The potential of calcium channel blockers in mitigating these seizure-induced cognitive impairments offers a promising therapeutic avenue [85, 86].“ This sentence needs to be rephrased. The articles cited here, numbers 86 and 87,  do not appear to address Ca channels or their inhibitors. The authors should explain specifically why they are citing these articles.

For articles listed in the  References, all authors should be included.

Author Response

Reviewer Comments

 

Response to Reviewer 1 comments

Major Comments

The authors answered almost all my questions and corrected the article according to most comments. However, they ignored some of the comments, or provided insufficient explanations, so the manuscript needs revision.

Detail Comments

In their response to the reviewer's question on why the effect of amlodipine is not seen under normal conditions, the authors state that they attribute the effect of amlodipine to its specific action under pathological conditions, where there is increased LTCCl expression. They do not elaborate on this explanation, which is somewhat illogical.   At the same time, however, they acknowledge that amlodipine also has an effect under physiological conditions but is "less noticeable". In the pictures, there is indeed this "less noticeable" effect occasionally visible, but it is never given as significant. The authors should therefore re-evaluate the effect of amlodipine in control experiments and/or at least comment on it in the text.

<Response: Thank you for your valuable feedback on our manuscript. We appreciate the opportunity to clarify our findings regarding the effects of amlodipine under normal physiological conditions.

In our study, we observed that amlodipine, known as an L-type calcium channel (LTCC) inhibitor, showed no significant effects under physiological conditions. This observation is particularly noteworthy as it underscores the specificity of amlodipine's action. In pathological states, characterized by altered LTCC expression as in epilepsy models, amlodipine demonstrated notable efficacy. However, in normal conditions, where LTCC activity remains within typical ranges, amlodipine does not exert significant modulatory effects.

This finding is critical as it suggests amlodipine's potential for targeted action in pathological states, without disturbing the normal physiological functioning of LTCCs. We believe this specificity could be highly beneficial in clinical settings, offering a therapeutic approach that minimizes unintended effects on normal cellular functions.

We have incorporated a discussion of these findings in our revised manuscript, emphasizing the importance of drug specificity and its implications for clinical therapy. We trust that this addition addresses your concern and enhances the manuscript's contribution to the understanding of amlodipine's pharmacological profile.>

The authors did not seem to understand, or did ignored, my comment to figures. Except figure 4, no images are still mentioned in the main text of Results.

<Response: Thank you for pointing out the oversight regarding the mention of figures in the main text of the Results section. We sincerely apologize for any confusion caused by this omission. In response to your comment, we have carefully reviewed the manuscript and have now explicitly referenced all relevant figures in the Results section. This includes the appropriate citation of each figure at the relevant points in the text, ensuring that the data and visual aids are properly integrated into the narrative of our results. This revision aims to enhance the clarity and comprehensiveness of our findings. We appreciate your attention to detail and your guidance in improving the quality of our manuscript. Thank you for helping us rectify this issue.>

Lines  738-739: „The potential of calcium channel blockers in mitigating these seizure-induced cognitive impairments offers a promising therapeutic avenue [85, 86].“ This sentence needs to be rephrased. The articles cited here, numbers 86 and 87,  do not appear to address Ca channels or their inhibitors. The authors should explain specifically why they are citing these articles.

<Response: Thank you for highlighting the need for clarification regarding the references cited in lines 738-739 of our manuscript. We agree with your observation and have taken steps to rectify this in our revised manuscript.

After reviewing the cited literature, we have amended the references to ensure they directly support the statements made. Specifically:

Line 742-744 Revision: We cited Ge (2020, PMID: 32407277) to support the statement: “Our findings suggest that chronic alterations in calcium channel function and expression, as a result of prolonged or repeated seizures, could contribute to the long-term cognitive deficits often observed in chronic epilepsy.” This reference provides evidence linking calcium channel function and cognitive deficits in epilepsy.

Line 745-747 Revision: For the sentence “The potential of calcium channel blockers in mitigating these seizure-induced cognitive impairments offers a promising therapeutic avenue,” we replaced the previous reference with Tan (2020, PMID: 32337274). This article directly discusses the role of calcium channel blockers in addressing cognitive impairments following seizures, thereby appropriately supporting our statement.

We have removed the reference to Malik (2022, PMID: 35976074) from this section, as upon re-evaluation, we agree that it does not directly support the discussed concept of calcium channel blockers in mitigating cognitive impairments due to seizures.

We believe these amendments provide a more accurate and relevant context to our discussion and appreciate your assistance in enhancing the accuracy of our manuscript.>

For articles listed in the References, all authors should be included.

<Response: Thank you for the important point of the reviewer and we found a problem where not all authors of the existing references were written and recorded all authors in the references. We changed it.>

Author Response File: Author Response.pdf

Reviewer 2 Report

The authors have addressed all of my comments accordingly.

The authors have addressed all of my comments accordingly.

Author Response

We appreciate this reviewer's comments.