Effect of Near-Infrared Pre-Irradiation on Irreversible Electroporation Treatment of Rat Gastric Tissues

Round 1

Reviewer 1 Report

Reviewer comments

 

 Dear Bora Keum

 

corresponding author

Applied Sciences

Manuscript ID: applsci-2574576

Type of manuscript: Article

Title: Effect of near-infrared pre-irradiation on irreversible electroporation treatment of rat gastric tissues

 

 

Major comments

There are some methodological defects in this paper.

The authors should add significant figures for the experimental procedures.

The authors should clarify how the dissection of rats (males or females) was carried out with photos.

The authors should clarify why they carried out the experimental procedures on the stomach.

 

The authors should provide why they prefer “Numerical analysis for electric field distribution” and “Numerical analysis for temperature distribution”. Provide Reference for these techniques.

 

Due to the large number of abbreviations, please write first in full name.

 

What mechanism？ Authors should give specific mechanism pathways of biological processes.

An obvious critical comment is the poor English language level in terms of syntax, punctuation, and grammatical errors (for example, put articles (a, an, and the) correctly.

A native English speaker should edit the manuscript text carefully. Since English is not our mother tongue, it will be better if the authors could check the manuscript again to remove some typos and syntax.

 

 

Author Response

1. There are some methodological defects in this paper.
-> We revised the whole methods and materials.

2. The authors should add significant figures for the experimental procedures.
-> We revised the experimental procedure in the Figure 1 in more detail. 

3. The authors should clarify how the dissection of rats (males or females) was carried out with photos.
-> We added more detailed rat dissection to the experimental procedures in Figure 1. 

4. The authors should clarify why they carried out the experimental procedures on the stomach.
-> We added the following in the Introduction section. (line 87-88)
“To validate this proposition, we turned to rat gastric tissues for our investigations, considering their accessibility for electroporation and NIR via endoscopic means.”

5. The authors should provide why they prefer “Numerical analysis for electric field distribution” and “Numerical analysis for temperature distribution”. Provide Reference for these techniques.
-> We added a relevant reference. 
Sabrina N.C.; Melvin F.L.; Brittanie P.; Nastaran A.; Yukitaka K.; Josefa G.; Sofie S.; Sean C.T.; Jonathan H.; Scott V.; Rafael V.D.; John H.R. High-frequency irreversible electroporation improves survival and immune cell infiltration in rodents with malignant gliomas. Front Oncol. 2023, 13, 1171278.

6. Due to the large number of abbreviations, please write first in full name.
->  We used the full name before first abbreviations. 

7. What mechanism. Authors should give specific mechanism pathways of biological processes.
-> We added the mechanisms in the discussion. 
“Apoptosis, a programmed cell death, typically manifests through unique signaling pathways [31]. Bcl-xl, associated with BCL2-like 1 gene, acts as an anti-apoptotic protein by preventing mitochondrial content release, such as cytochrome c. The p21 protein regulates cell cycle progression], while SOD2 facilitates electron transport activity, converting oxidative phosphorylation byproducts to hydrogen peroxide and diatomic oxygen. Among these proteins, SOD2 expression was observed to increase post IRE treatment [33,34]. In addition, p21 protein expression was enhanced by IRE treatment [32], through western blotting. However, Bcl-xl expression was reduced by IRE treatment [35]. In our experiment, Bcl-xl expression decreased with IRE but increased further with NIRE treatment compared to IRE. Bcl-xl, a member of the Bcl-2 family, is vital in apoptosis regulation [36], and its altered expression in many cancers helps evade apoptosis [37]. Conversely, activation of BCL2-associated x protein (BAX) and BCL2-antagonist-killer (BAK) can initiate apoptotic events through cytochrome c release. The enhanced Bcl-xl expression in NIRE-treated samples, compared to controls, suggests a potential increase in apoptotic signals. Further studies on expression of Bcl-xl due to NIRE treatment are needed. Moreover, p21 activation was more prominent in NIRE than IRE-treated samples, aligning with previous findings that link p21 activation to DNA damage and subsequent apoptosis [38-40]. In addition, diminished SOD2 expression across experimental groups relative to controls implies that ROS production might be influenced directly by either treatment. Given SOD2’s role in safeguarding against cell death, our findings suggest NIRE could potentiate apoptosis alongside IRE. Investigating Bcl-xl, p21, and SOD2 expressions in depth, especially concerning their roles in apoptosis, remains crucial.”

8. We carried out English revision on the manuscript. 
-> We performed English revision thorough the manuscript.

 

Author Response File: Author Response.docx

Reviewer 2 Report

I have reviewed the manuscript of "Effect of near-infrared pre-irradiation on irreversible electroporation treatment of rat gastric tissues", it was very interesting to read and I decided to provide Major Revision with essential comments to improve the manuscript.

-       Entire abstract should be belongs to what your entire result and it should be brief, short and indicate all the results clearly. Should not leave any obtained results.

-       Introduction, please provide more details about mentioned proteins.

-       The brand of the device and kit for western band and also the dilution of secondary antibodies should be provided.

-       The authors try to include better quality of Figure 1 and 2.

-       This method was designed for cancer treatment and the author should perform this method in rat with induced gastric cancer.  

-       In Figure 5, Y axis was written actine. Please edit to actin.

-       In Figure 5 B, author should statistically compare all groups together and in text added.

-       To confirm western blot assay, real time PCR should be performed.

-       To improve the discussion section, should cite additional papers. Author should be mentioned recent references for discussion of all the results.

-       Conclusion should be mentioned the concluded final result and expanded. 

 

Author Response

1. Entire abstract should be belongs to what your entire result and it should be brief, short and indicate all the results clearly. Should not leave any obtained results.
-> We revised the abstract entirely.
“Irreversible electroporation (IRE) is a recognized ablation technique that induces apoptosis via potent electric fields. Nonetheless, the heterogeneity of biological tissues often results in incon-sistent treatment outcomes, leaving residual viable cells and leading to potential relapse. To ad-dress this, previous strategies incorporated chemical enhancers to IRE, but these faced limitations such as limited tissue diffusion and hyperpigmentation. In this study, we explore the synergistic application of Near-Infrared (NIR) irradiation with IRE. Using an in vivo rat gastric tissue model, we pre-irradiated samples with NIR at 3 J/cm2 prior to IRE. The combined treatment, termed NIRE, produced a change in tissue impedance of 13.5 Ohm compared to IRE alone, indicating NIR's potential in modulating tissue electrical properties. Subsequent histopathological and mo-lecular assessments revealed a 1.12-fold increase in apoptosis for NIRE over IRE. Notably, apop-tosis-related protein BCL and p21 exhibited a 1.24-fold and 1.29-fold overexpression following NIRE treatment respectively, emphasizing NIRE's enhanced apoptotic activation. In essence, our findings underscore the augmented therapeutic efficacy of IRE when complemented with NIR, presenting a promising avenue for bolstering treatment outcomes in tissue ablation.”

2. Introduction, please provide more details about mentioned proteins.
-> We added the introduced proteins in the introduction
“To further elucidate their combined effects at the protein level, we introduced and as-sessed apoptotic-related proteins such as B-cell lymphoma-extralarge (Bcl-xl), cy-clin-dependent kinase inhibitor (p21), and superoxide dismutase 2 (SOD2).”

3. The brand of the device and kit for western band and also the dilution of secondary antibodies should be provided.
-> We addend information on kits for western blot and the dilution of secondary antibodies. (Line208-217)
“The membrane was then incubated with primary antibodies against B-cell lympho-ma-extralarge (Bcl-xl) (Invitrogen, Thermo Fisher Scientific, USA), cyclin-dependent kinase inhibitor (p21) (Invitrogen, Thermo Fisher Scientific, USA), and superoxide dismutase 2 (SOD2) (Invitrogen, Thermo Fisher Scientific, USA) at a 1:1,000 dilution overnight at 4℃. The following day, the membrane was incubated with secondary an-tibodies (m-IgGk BP-HRP; IgG-HRP; Chicken anti-goat igG-HRP, Santa Cruz, Santa Cruz Biotechnology Inc., USA) at a dilution of 1:5,000 for 1.5 h at room temperature. Protein bands were visualized using an enhanced chemiluminescence (ECL) substrate (SuperSignal, ThermoFisher Scientific, USA). Densitometric values of the proteins were quantified, normalized to β-actin (Santa Cruz, Santa Cruz Biotechnology Inc., USA), and represented as histograms.”

4. The authors try to include better quality of Figure 1 and 2.
-> We qualified better Figure 1 and 2. 

5. This method was designed for cancer treatment and the author should perform this method in rat with induced gastric cancer.  
-> Your indication is right. We confined to feasibility NIRE effect on normal gastric tissues.

6. In Figure 5, Y axis was written actine. Please edit to actin.
-> We did as the following.

7. In Figure 5 B, author should statistically compare all groups together and in text added.
-> We evaluated statistical data among groups. 

8. To confirm western blot assay, real time PCR should be performed.
-> Your indication is right. The PCR have to be analyzed following western blot assay. We are going to conduct further study.  

9. To improve the discussion section, should cite additional papers. Author should be mentioned recent references for discussion of all the results.
-> We cited recently references in the discussion. 

10. Conclusion should be mentioned the concluded final result and expanded.
->  We revised the conclusion as following: 
“Our findings demonstrate that NIRE treatment resulted in a tissue impedance change of 13.5 Ohm relative to IRE treatment. In terms of apoptosis induction, NIRE led to an increase of 1.12 times in comparison to IRE. Furthermore, the expression of the apoptosis-associated protein BCL was amplified by 1.24 times, p21 protein by 1.29 times, and SOD2 exhibited a 0.93-fold expression in NIRE-treated tissues as compared to those treated by IRE. This enhanced response can be attributed to the interactions between NIR and cells, which seem to modulate cellular metabolism prior to IRE treatment. Importantly, we observed no adverse or toxic effects from NIR exposure on the tissue. These findings hold promise for augmenting the apoptotic efficacy of IRE in therapeutic settings.”

 

Author Response File: Author Response.docx

Reviewer 3 Report

Han Jo Jeon and co-workers have submitted an article titled “Effect of near-infrared pre-irradiation on irreversible electroporation treatment of rat gastric tissues”. The work is interesting and has potential application. There are the following concerns that need to be addressed before publication.

 

1. Authors should highlight the novelty in view of future perspectives in the abstract.
2. Authors should elaborate the mechanism of action.
3. Authors should explain toxicity factor of direct irradiation.
4. Conclusions should be elaborated with respect to quantitative outcomes and future perspectives.
5. The study has limitations, there should be more evidences for apoptotic proteins study.

Comments for author File: Comments.pdf

Some minor mistakes can be improved during revision.

Author Response

1. Authors should highlight the novelty in view of future perspectives in the abstract.
-> We revised the abstract. 
“Irreversible electroporation (IRE) is a recognized ablation technique that induces apoptosis via potent electric fields. Nonetheless, the heterogeneity of biological tissues often results in incon-sistent treatment outcomes, leaving residual viable cells and leading to potential relapse. To ad-dress this, previous strategies incorporated chemical enhancers to IRE, but these faced limitations such as limited tissue diffusion and hyperpigmentation. In this study, we explore the synergistic application of Near-Infrared (NIR) irradiation with IRE. Using an in vivo rat gastric tissue model, we pre-irradiated samples with NIR at 3 J/cm2 prior to IRE. The combined treatment, termed NIRE, produced a change in tissue impedance of 13.5 Ohm compared to IRE alone, indicating NIR's potential in modulating tissue electrical properties. Subsequent histopathological and mo-lecular assessments revealed a 1.12-fold increase in apoptosis for NIRE over IRE. Notably, apop-tosis-related protein BCL and p21 exhibited a 1.24-fold and 1.29-fold overexpression following NIRE treatment respectively, emphasizing NIRE's enhanced apoptotic activation. In essence, our findings underscore the augmented therapeutic efficacy of IRE when complemented with NIR, presenting a promising avenue for bolstering treatment outcomes in tissue ablation.”

2. Authors should elaborate the mechanism of action.
-> We added the mechanisms in the discussion. 
“Apoptosis, a programmed cell death, typically manifests through unique signaling pathways [31]. Bcl-xl, associated with BCL2-like 1 gene, acts as an anti-apoptotic pro-tein by preventing mitochondrial content release, such as cytochrome c . The p21 protein regulates cell cycle progression, while SOD2 facilitates electron transport activity, converting oxidative phosphorylation byproducts to hydrogen peroxide and diatomic oxygen. Among these proteins, SOD2 expression was observed to in-crease post IRE treatment [33,34]. In addition, p21 protein expression was enhanced by IRE treatment [32], through western blotting. However, Bcl-xl expression was reduced by IRE treatment [35]. In our experiment, Bcl-xl expression decreased with IRE but in-creased further with NIRE treatment compared to IRE. Bcl-xl, a member of the Bcl-2 family, is vital in apoptosis regulation [36], and its altered expression in many cancers helps evade apoptosis [37]. Conversely, activation of BCL2-associated x protein (BAX) and BCL2-antagonist-killer (BAK) can initiate apoptotic events through cytochrome c release. The enhanced Bcl-xl expression in NIRE-treated samples, compared to controls, suggests a potential increase in apoptotic signals. Further studies on expres-sion of Bcl-xl due to NIRE treatment are needed. Moreover, p21 activation was more prominent in NIRE than IRE-treated samples, aligning with previous findings that link p21 activation to DNA damage and subsequent apoptosis [38-40]. In addition, dimin-ished SOD2 expression across experimental groups relative to controls implies that ROS production might be influenced directly by either treatment. Given SOD2’s role in safeguarding against cell death, our findings suggest NIRE could potentiate apoptosis alongside IRE. Investigating Bcl-xl, p21, and SOD2 expressions in depth, es-pecially concerning their roles in apoptosis, remains crucial.”

3. Authors should explain toxicity factor of direct irradiation.
-> We consider your statement about which NIR irradiation may cause thermal effects. However, 3 J/cm2 that we used in this study, is too low to happen thermal effects. So, we added this in the discussion section of the manuscript as following (331-333)
“Despite the thermal effects suggested by another study, temperature may play a minor role in influencing electrical impedance.”

-> It is added in the conclusions.
“Importantly, we observed no adverse or toxic effects from NIR exposure on the tissue.”

4. Conclusions should be elaborated with respect to quantitative outcomes and future perspectives.
-> We revised the conclusion section. 
“Our findings demonstrate that NIRE treatment resulted in a tissue impedance change of 13.5 Ohm relative to IRE treatment. In terms of apoptosis induction, NIRE led to an increase of 1.12 times in comparison to IRE. Furthermore, the expression of the apoptosis-associated protein BCL was amplified by 1.24 times, p21 protein by 1.29 times, and SOD2 exhibited a 0.93-fold expression in NIRE-treated tissues as compared to those treated by IRE. This enhanced response can be attributed to the interactions between NIR and cells, which seem to modulate cellular metabolism prior to IRE treatment. Importantly, we observed no adverse or toxic effects from NIR exposure on the tissue. These findings hold promise for augmenting the apoptotic efficacy of IRE in therapeutic settings.”

5. The study has limitations, there should be more evidences for apoptotic proteins study.
-> Your indication is right. We confined proteins to BCL, p21, and SOD2.

6. Some minor mistakes can be improved during revision.
->  We revised the thorough manuscript.  

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Dear Bora Keum

 

corresponding author

Applied Sciences

Manuscript ID: applsci-2574576-peer-review-v2

Type of manuscript: Article

Title: Effect of near-infrared pre-irradiation on irreversible electroporation treatment of rat gastric tissues

 The authors provided the reviewed major comments.

No comments

 

Well done.

Reviewer 2 Report

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