Effect of Photo-Mediated Ultrasound Therapy on Nitric Oxide and Prostacyclin from Endothelial Cells

Round 1

Reviewer 1 Report

The authors show biphasic effects of photo-mediated ultrasound on the release of nitric oxide and prostacyclin from endothelial cells, with alteration of endothelial cells functions with high negative pressure linked to cavitation phenomenom, and enhanced when laser pulses are added.

Some questions on the methods :

• accuracy of the endothelial cells model, i.e. chorioretinal endothelial cells from Rhesus monkey, is it a valuable model for the study on human diseases? and in particular in ophtalmology?  We know that endothelial cells exhibit various functions depending on their localization in the body, e.g. different when derived from artery, vein or microvasculature, or a cell line that can loose some functions; this point must be discussed as well as the use of this model for studies in ophtalmology as it applied.
• The method for nitric oxide measurement (Griess method) is not known as a very specific method (also measures nitrate and nitrite); not discussed or referenced
• The method for prostacyclin measurement (immuno-assay), nothing about its specificity; also not discussed and referenced

 

Author Response

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Reviewer 2 Report

This is a nice study providing details on how to develop a vessels model and study the effects of PUT.

It would be interesting to add more background knowledge: what other similar models exist? what did previous studies show? in which domains PUT is therapeutically used? 

Author Response

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Reviewer 3 Report

This looks like a solid study with good experimental design. It should be beneficial and interesting to those in the fields of US and photodynamic therapy.

There are only a few issues to address:

-the authors should discuss the expected depth of ultrasound (US) and laser irradiation in skin. since the study is based on epithelial cells, the big picture could likely treatment in outpatient settings. would the system be suitable for treatment below the skin's surface?

-the 2 MPa pressure trace is missing from figure 7

-trends are not so clear; in at least 3 cases the 1.5 MPa pressure or 15 mJ/cm² laser irradiation trials produced results that are inconsistent with the expected trend. there may be some critical point at which the trends reverse due to excessive cell death, cavitation heating, or some other effect. the authors should elaborate on the possibilities in the discussion.

-cavitation, especially at lower US frequencies can result in significant heating. the authors should mention any controls they performed to account for heating or to reinforce the lack of it at these frequencies and pressures.

Author Response

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Reviewer 4 Report

Manuscript ID applsci-1598085 describes the impact of PUT on endothelial cell Function, notably their capacity to produce NO and PGI2.

Although this manuscript is interesting, the following points should be addressed. 

• The introduction section is too long. The authors in several sections have mentioned their aim of the study. I suggest focusing on the problematics and previous advancements and then elaborating the exact aim of the study in a single section.
• I am not convinced if culturing the cell in static conditions can significantly reduce the chance of contamination. They should have performed their experiment in underflow conditions as well. Please refer to the following article: PMID: 34309615. Moreover, although the static condition can avoid shear stress but the real in vivo situation the shear stress is present and plays an important role. 
• For nitrate reduction and prostacyclin assays, which manufacturers were used?
• Although NO and PGI2 are some important factors in endothelial cell biology, however, they are far from being the only elements to be measured for endothelial dysfunction. The authors should have correctly characterized endothelial essential markers and other biomarkers related to their cells damage and dysfunction. Please refer to the following articles: 33397378, 32546175. 

Minor comments

* Please avoid repeating ''could'' in line 308. 

Author Response

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Round 2

Reviewer 4 Report

Although only by explanation and not further experiments, the authors have addressed some of my concerns. 

I have no further comments.