Central Autonomic Network Regions and Hypertension: Unveiling Sympathetic Activation and Genetic Therapeutic Perspectives

Round 1

Reviewer 1 Report

In their work entitled “Central Autonomic Network Regions and Hypertension: Unveiling Sympathetic Activation and Genetic Therapeutic Perspectives” Geraldes and colleagues investigate the role of specific brain regions namely the lateral para-14 brachial nucleus (LPBN), Kolliker-fuse nucleus (KF), and periductal grey matter (PAG) in regulating blood pressure using genetic modification (lentiviral vectors). This research highlight the role of LPBN in hypertension and may contribute to knowledge required for novel treatment approaches to hypertension.

 

Major Comments:

1.      No major comments. Scientifically sound.

Minor Comments:

1.      Image resolution of figure 1-5 is low. The use of higher resolution versions of the figure would be beneficial to the reader.

2.      Figure 1 please add a figure key indicating content represented by the grey and black lines respectively.

3.      Page 11 line 370: Some text may accidentally be highlighted in grey.

Author Response

Thank you to the reviewers for your useful and constructive suggestions. We have revised the present manuscript and considered the reviewers suggestions and comments, since these changes will produce an article that better serves you and our readers.

We hope that this revised manuscript will be subject to satisfactory responses to the reviewers´ comments.

The answers to their specific comments/suggestions are as follows.

Response to Reviewer 1 Comments

In their work entitled “Central Autonomic Network Regions and Hypertension: Unveiling Sympathetic Activation and Genetic Therapeutic Perspectives” Geraldes and colleagues investigate the role of specific brain regions namely the lateral para-14 brachial nucleus (LPBN), Kolliker-fuse nucleus (KF), and periductal grey matter (PAG) in regulating blood pressure using genetic modification (lentiviral vectors). This research highlight the role of LPBN in hypertension and may contribute to knowledge required for novel treatment approaches to hypertension.

Major Comments:

1. No major comments. Scientifically sound.

Minor Comments:

1. Image resolution of figure 1-5 is low. The use of higher resolution versions of the figure would be beneficial to the reader.

Thanks for your remark. We have improved the image resolution of figure 1-5.

2. Figure 1 please add a figure key indicating content represented by the grey and black lines respectively.

Sorry, it was an error in the copy and paste of the image. We added this information in the figure and in the figure legend.

3. Page 11 line 370: Some text may accidentally be highlighted in grey.

We already changed. Thank you.

Reviewer 2 Report

This manuscript presents a comprehensive investigation about how the brain, specifically the central autonomic network, plays a role in essential hypertension.

This work investigated on role of specific brain regions in regulating blood pressure in the context of hypertension, a prevalent cause of mortality. The brain regions studied include the Lateral Parabrachial Nucleus (LPBN), Kolliker-Fuse Nucleus (KF), and the Periductal Grey Matter (PAG). The use of lentiviral vectors to modulate the activity of these regions in hypertensive rats over 75 days and monitoring blood pressure, cardiac rate, reflex response, and heart rate variability present a systematic approach. The results provide insightful findings. For instance, reducing activity in the LPBN decreased sympathetic outflow which lowered both blood pressure and heart rate. Outcomes from the KF showed lowered sympathetic activity and chemoreflex variation alteration, but without the corresponding effect on blood pressure. Silencing of PAG did not significantly alter either blood pressure or sympathetic tone, but it did reduce the cardiac baroreflex gain. It suggests that both LPBN and KF neurons are instrumental in activating mechanisms controlling respiration and sympathetic outflows during chemoreceptor activation.

 

Major comments,

1 It would be beneficial to clarify how the work can contribute to new treatment approaches, particularly about genetic therapies such as adenovirus-based COVID-19 vaccines. This may seem disjointed as hypertension and COVID-19 are quite different pathologies.

2 Please give reference for the methods for evaluation of Baroreceptor Reflex and chemoreceptor reflex, as well as details in process. Do they have good enough precision?

3 What is Lentiviral? How it works? It is needed to show results that prove the lentiviral works in special regions. Are there any off-target effects of microinjections?

4 Figure 1. What are the two lines and points? Please label them.

5 To improve the manuscript, it would be helpful if the authors provided a more detailed discussion on the specific pathways and neural circuits through which the LPBN, PAG, and KF exert their effects on cardiovascular and respiratory functions. This would enhance the understanding of the underlying mechanisms and provide a stronger basis for future research directions.

6 The authors mention that regions like the PAG, LPBN, and KF vary in their control of sympathetic activity. Please describe how each region impacts hypertension distinctly.

7 Are those lentiviral can regulated LPBN, KF, PAG really? why different response shown in distinct regions with same lentiviral?

N/A

Author Response

Thank you to the reviewer for your useful and constructive suggestions. We have revised the present manuscript and considered the reviewer suggestions and comments, since these changes will produce an article that better serves you and our readers.

We hope that this revised manuscript will be subject to satisfactory responses to the reviewers´ comments.

The answers to their specific comments/suggestions are as follows.

Review Report (Reviewer 2)

This manuscript presents a comprehensive investigation about how the brain, specifically the central autonomic network, plays a role in essential hypertension.

This work investigated on role of specific brain regions in regulating blood pressure in the context of hypertension, a prevalent cause of mortality. The brain regions studied include the Lateral Parabrachial Nucleus (LPBN), Kolliker-Fuse Nucleus (KF), and the Periductal Grey Matter (PAG). The use of lentiviral vectors to modulate the activity of these regions in hypertensive rats over 75 days and monitoring blood pressure, cardiac rate, reflex response, and heart rate variability present a systematic approach. The results provide insightful findings. For instance, reducing activity in the LPBN decreased sympathetic outflow which lowered both blood pressure and heart rate. Outcomes from the KF showed lowered sympathetic activity and chemoreflex variation alteration, but without the corresponding effect on blood pressure. Silencing of PAG did not significantly alter either blood pressure or sympathetic tone, but it did reduce the cardiac baroreflex gain. It suggests that both LPBN and KF neurons are instrumental in activating mechanisms controlling respiration and sympathetic outflows during chemoreceptor activation.

 Major comments,

1 It would be beneficial to clarify how the work can contribute to new treatment approaches, particularly about genetic therapies such as adenovirus-based COVID-19 vaccines. This may seem disjointed as hypertension and COVID-19 are quite different pathologies.

Yes, you are right. But we just added this information since these findings have broader implications for the development of novel treatment approaches, including genetic therapies like adenovirus-based COVID-19 vaccines. In the context of COVID-19, genetic therapies, such as adenovirus-based vaccines, have gained attention for their potential to combat the virus. These vaccines use modified adenoviruses to deliver genetic material, an approach similar to our study. By applying the knowledge gained from our study to genetic therapies, it may be possible to explore innovative treatment approaches for COVID-19, considering the potential role of the LPBN and KF in regulating respiratory and sympathetic responses, which can be relevant in managing severe cases of COVID-19 with cardiovascular complications. However, further research and clinical studies would be necessary to validate and apply these findings to COVID-19 treatments specifically.

Nevertheless, after reading your comment, we agree with you and we removed this sentence on Covid from the conclusion.

2 Please give reference for the methods for evaluation of Baroreceptor Reflex and chemoreceptor reflex, as well as details in process. Do they have good enough precision?

Thank you for your comment. To determine if the methods have good enough precision, several factors need to be considered. However, the methods used in our study for the evaluation of the Baroreceptor Reflex and Chemoreceptor Reflex are frequently used, reproducible and accepted in physiological studies.

The baroreflex evaluation involves the intravenous injection of phenylephrine, which elicits an immediate and progressive increase in mean blood pressure accompanied by a consequent decrease in heart rate. Phenylephrine bolus injection is an established technique to measure baroreflex function. The magnitude of the response induced by phenylephrine is measured by the ratio of ΔHR/ΔBP. 

The chemoreceptor reflex (ChR) was assessed by quantifying the change in respiratory rate (∆ChR) following lobeline stimulation. The respiratory rate (RespR) was determined based on tracheal pressure measurements taken before and after lobeline administration, and the calculation was performed using the following formula: ∆ChR = RespR_lobeline - RespR_basal.

As suggested, we added the reference for evaluation of Baroreceptor Reflex and chemoreceptor reflex in the methods section.

3 What is Lentiviral? How it works? It is needed to show results that prove the lentiviral works in special regions. Are there any off-target effects of microinjections?

A lentivirus is a type of retrovirus and is known for its ability to infect the genome of the host cell and integrate its genetic material into it. These viruses have a slower replication rate compared to other retroviruses. Lentiviruses are enveloped viruses, meaning they have an outer lipid membrane surrounding their genetic material. Their genetic material consists of single-stranded RNA that is converted to double-stranded DNA during the viral replication process.

Therefore, lentiviruses are of great interest to researchers because they can introduce genetic material into the genome of the host cell. This property has been exploited to develop lentiviral vectors. These are modified lentiviruses used in gene therapy and gene research. Lentiviral vectors can be used to introduce genes of interest into cells or to modify specific genes in the host cell's DNA, offering promising potential for the treatment of genetic disorders and other medical conditions.

Regarding how it works, the lentivirus first attaches to specific receptors on the surface of the host cell. Once attached, the virus fuses with the host cell membrane, releasing its contents into the cell. Once inside the host cell, the viral RNA is reverse transcribed by an enzyme called reverse transcriptase. This process converts the viral RNA into double-stranded DNA (Reverse Transcription). Then the newly formed viral DNA is transported into the cell nucleus, where it integrates into the host cell's DNA, allowing the viral genetic material to become a permanent part of the host cell's genome. The integrated viral DNA is transcribed and translated by the host cell's machinery, producing new viral RNA and viral proteins that assemble into new viral particles, which then bud off from the host cell, acquiring an envelope derived from the host cell membrane. These new viral particles can then go on to infect other cells and continue the viral replication cycle.

As stated in 2.4.2, the viral vector construction and validation was based on previous studies [19-21]. Briefly, LVV-hKir2.1 is a mix of LV-TREtight-Kir-cIRES-GFP 5.4×109 IU and LV-Syn-Eff-G4BS-Syn-Tetoff 6.2×109 IU (ratio 1:4), which expresses eGFP as well as human inwardly rectifying potassium channels of the hKir2.1 type in neuronal cells. LVVeGFP, used for the SHAM group (used as a control group to prevent bias and involved a procedure that mimics the experimental intervention without delivering the active treatment), was a mixture of LVTREtight-GFP 5.7×109 IU) and LV-Syn-Eff-G4BS-Syn-Tetoff 6.2×109 IU in a 1:4 ratio. This binary system expresses eGFP.

Validation of transduction and transgene expression efficacy was performed as described previously and included mRNA expression, immunocytochemical, and electrophysiological data [19-21]. After the acute experiment, the animal’s brains were removed and coronal sections were cut and mounted on slides. Using an epifluorescence microscope, eGFP-labelled regions were identified and plotted on standardised sections of the Paxinos and Watson atlas. In order to minimize the error, the stereotaxic coordinates were tested in 3 animals, in our frame, before starting this group of experiments. We did not study the off-target effects of microinjections, because all microinjected areas were evaluated, confirming the microinjection site.

4 Figure 1. What are the two lines and points? Please label them.

Sorry, it was an error in the copy and paste of the image. Thank you for your observation.

5 To improve the manuscript, it would be helpful if the authors provided a more detailed discussion on the specific pathways and neural circuits through which the LPBN, PAG, and KF exert their effects on cardiovascular and respiratory functions. This would enhance the understanding of the underlying mechanisms and provide a stronger basis for future research directions.

As suggested, we added the specific pathways and neural circuits through which the LPBN, PAG, and KF exert their effects on cardiovascular and respiratory functions in the discussion section: “The LPBN, KF nucleus, and PAG are brain regions that form interconnected neural circuits involved in the regulation of cardiovascular and respiratory functions. They receive inputs from various brain centers and sensory information from the periphery, such as baroreceptor and chemoreceptor inputs, integrating this information to modulate autonomic outflow and respiratory patterns”.

And we had already added this information to the discussion:

“PAG columns can modulate cardiac sympathetic function through indirect pathways involving sympathetic premotor neurones found at specific sites in the hypothalamus, midbrain, pons, and medulla oblongata, modulating various physiological functions, including cardiovascular and respiratory activities. Additionally, the major outflow of the PAG terminates in the bulbospinal regions of the RVLM. Therefore, PAG silencing results in a decrease in sympathetic activity, which activates adjacent sympathetic areas via its projections [28, 29, 30].”

“The LPBN can relay autonomic information to other structures, which is crucial for the regulation of autonomic function, enabling it to regulate sympathetic and parasympathetic outflow to the cardiovascular system and to promote changes in heart rate and peripheral vascular resistance. The LPBN projects to the RVLM, and, according to Kubo et al. LPBN pressor site neurones mediate the cholinergic inputs responsible for RVLM pressor responses [33]. Consequently, LPBN-reduced activity could act on silencing sympathetic pre-motor neurones by tackling the RVLM and, hence, indirectly, IML neurones or by directly working on them."

“Located in the brainstem, the KF is a pontine nucleus that regulates respiration, by modulating respiratory rate and pattern, through its projections to the core respiratory nuclei in the brainstem, including the pre-Bötzinger complex, lateral parafacial nucleus, Bötzinger complex, and rostral ventral respiratory group projections [40]. Thus, the significant variation in chemoreceptor function was not surprising because of the decreased sympathetic activity evoked by KF modulation of excitability and the absence of cardiovascular responses. The decrease in neuronal excitability in the KF region showed that the affected neurones could be part of the central mechanisms involved in the chemosensory control of the sympathetic and respiratory chemoreflex.”

6 The authors mention that regions like the PAG, LPBN, and KF vary in their control of sympathetic activity. Please describe how each region impacts hypertension distinctly.

The LPBN, KF and PAG are interconnected brain regions that play distinct roles in regulating sympathetic activity and, consequently, impact hypertension differently: the LPBN has a significant influence on hypertension-related sympathetic activation. The LPBN's decreased activity reduces sympathetic outflow, leading to decreased blood pressure and heart rate, thus impacting hypertension positively. The KF nucleus primarily affects chemoreflex modulation, whereas its direct impact on blood pressure regulation appears to be limited. The PAG's role in hypertension is complex, involving modulation of sympathetic and parasympathetic outflow, and may influence specific aspects of cardiovascular function. Activation of the PAG can lead to changes in sympathetic activity, which can affect heart rate and blood pressure responses. However, in the present study, silencing the PAG did not have a significant impact on blood pressure or sympathetic tone but decreased cardiac baroreflex gain, indicating a more complex role in cardiovascular regulation.

7 Are those lentiviral can regulated LPBN, KF, PAG really? why different response shown in distinct regions with same lentiviral?

Thank you for sharing your observation. It is important to clarify that lentiviral vector used were employed to introduce genetic material into neuronal cells in specific brain regions. They do not directly regulate or control the function of brain regions like the Lateral Parabrachial nucleus (LPBN), Kolliker-Fuse nucleus (KF), or Periaqueductal Grey Matter (PAG). Instead, they deliver genetic material, namely hKir2.1, which decreased the activity of neuronal cells. By genetically modulating cell excitability to decrease the activity of these areas, we assessed long-term effects on blood pressure, peripheral sympathetic tone, and cardiovascular reflex responses in an animal model of essential hypertension. The different responses observed in distinct brain regions when using the same lentiviral vector can be attributed to the reduction in the activity of these brain areas due to the decrease in the action potential of neuronal cells.

Reviewer 3 Report

This is a very nicely performed experiment on hypertensive rats that were administrated  lentoviral vectors microinjections.

The Authors focused on the three brain regions, including LPBN, KF and PAG responsible for blood pressure control. They found that particularly targetting LPBN resulted in blood pressure decrease, as well as heart rate decrease. While, PAG and KF showed no associations with blood pressure lowering. In conclusion, genetic modulations with lentovirus might facilitate therapy of ( perhaps resistant) hypertension.

Introduction, Methods, results' presenation are clearly presented. Discussion is comprehensive and well-written.

I have only some minor remarks.

1. Figure 1 needs explanation for bars, e.g pale grey stands for control group, dark grey - study group

2. Conclusions. I do not think that the last sentence on Covid is necessary. The Authors did not study this issue 

3. Please explain term SHAM , spontaneously hypertensive animal model rats? in the text when it occurs for the first time.

4. Discussion. Please consider to enhance the discussion with more up-to-date papers, including novel studies on genetic modifications of blood pressure, like : Ohara H, et al. Genetic modifications of alter blood pressure level. Doi: https://doi.org/10.3390/biomedicines10081855 and Jun S, et al. Neuroscience biulletin 2023, january. https://doi.org/10.1007/s12264-022-01008-3 

Author Response

Thank you to the reviewers for your useful and constructive suggestions. We have revised the present manuscript and considered the reviewers suggestions and comments, since these changes will produce an article that better serves you and our readers.

We hope that this revised manuscript will be subject to satisfactory responses to the reviewers´ comments.

The answers to their specific comments/suggestions are as follows.

Review Report (Reviewer 3)

This is a very nicely performed experiment on hypertensive rats that were administrated  lentoviral vectors microinjections.

The Authors focused on the three brain regions, including LPBN, KF and PAG responsible for blood pressure control. They found that particularly targetting LPBN resulted in blood pressure decrease, as well as heart rate decrease. While, PAG and KF showed no associations with blood pressure lowering. In conclusion, genetic modulations with lentovirus might facilitate therapy of ( perhaps resistant) hypertension.

Introduction, Methods, results' presenation are clearly presented. Discussion is comprehensive and well-written.

I have only some minor remarks.

1. Figure 1 needs explanation for bars, e.g pale grey stands for control group, dark grey - study group

Sorry, it was an error in the copy and paste of the image. We have already added the caption in the figure. Thank you for your observation.

2. I do not think that the last sentence on Covid is necessary. The Authors did not study this issue 

Yes, you are right. But we just added this information since these findings have broader implications for the development of novel treatment approaches, including genetic therapies like adenovirus-based COVID-19 vaccines. In the context of COVID-19, genetic therapies, such as adenovirus-based vaccines, have gained attention for their potential to combat the virus. These vaccines use modified adenoviruses to deliver genetic material, an approach similar to our study. By applying the knowledge gained from our study to genetic therapies, it may be possible to explore innovative treatment approaches for COVID-19, considering the potential role of the LPBN and KF in regulating respiratory and sympathetic responses, which can be relevant in managing severe cases of COVID-19 with cardiovascular complications. However, further research and clinical studies would be necessary to validate and apply these findings to COVID-19 treatments specifically.

Nevertheless, after reading your comment, we agree with you and we removed this sentence on Covid from the conclusion.  

3. Please explain term SHAM , spontaneously hypertensive animal model rats? in the text when it occurs for the first time.

Thank you for your observation. The SHR are the spontaneously hypertensive animal model rats. The sham group is a type of control group used in studies where blinding is necessary. Our sham group was used as a control to prevent bias and involved a procedure that mimics the experimental intervention (craniotomy) without delivering the active treatment. In the SHAM group we delivered the LVVeGFP, in the brain regions, but this lentiviral vector only expresses eGFP. We added this information in the methods section.

4. Discussion. Please consider to enhance the discussion with more up-to-date papers, including novel studies on genetic modifications of blood pressure, like : Ohara H, et al. Genetic modifications of alter blood pressure level. Doi: https://doi.org/10.3390/biomedicines10081855 and Jun S, et al. Neuroscience biulletin 2023, january. https://doi.org/10.1007/s12264-022-01008-3 

As suggested, we have included these two papers in the discussion.

Round 2

Reviewer 2 Report

Although authors declared: "Using an epifluorescence microscope, eGFP-labelled regions were identified and plotted on standardised sections of the Paxinos and Watson atlas.", 

the images or hKir2.1 expression are needed to present the specificity.

Author Response

Dear Reviewer,

We sincerely appreciate your valuable feedback on our manuscript. As described in the Methods, we identified the microinjected areas using the Paxinos and Watson atlas and ensured the integrity and accuracy of our study. As suggested by you, we added an image on the Paxinos and Watson atlas showing the microinjected areas and the eGFP expression in the three different areas.

We sincerely hope that this revised manuscript adequately addresses the concerns raised.

Thank you for your time and dedication in reviewing our manuscript.

Sincerely,

Vera Geraldes

 

Author Response File: Author Response.pdf

Round 3

Reviewer 2 Report

Authors had adressed all my issues.