Study of pulsed electrostatic field (PESF) in the perfusion of peripheral tissues: Microangiopathy, nutrition and quality of perceiver life.

Microangiopathy compromises the structural and functional integrity of organs and tissues in patients with type II diabetes mellitus (T2DM) negatively affecting the perceived quality of life. Nitric oxide (NO) is a multifunctional signalling molecule, acting as a vasodilator, neurotransmitter, and modulator of inflammatory processes. Patients with type II diabetes mellitus and chronic kidney disease, controlled from glycaemic status, were treated or not with pulsed electrostatic field (PESF) cycles to evaluate effect on the perfusion of peripheral tissues. Everyone was monitored for the metabolic profile, and we tested circulating NO with a commercial enzyme immunoassay kit. In addition, we tested the perceived quality of life of patients before/after a PESF cycle using a questionnaire. Patients treated with PESF were improved circulating NO levels, significant changes in systolic and diastolic blood pressure, heart rate and were more homogeneous for their metabolic profile. The questionnaire showed also a marked improvement in the perceived quality of life. The use of pulsed electrostatic fields has allowed us to observe an improvement in the metabolic, psychological, and clinical profile in patients with T2DM and chronic kidney disease whose pathological profile is strongly compromised.

Introduction

The prognosis for patients with type 2 diabetes mellitus (T2DM) has changed significantly over the course of a few decades thanks to the continuous technological and pharmacological innovations as a result of a fruitful collaboration between industry and scientific-healthcare world. Despite the progress in terms of survival, the perceived quality of life in old patients with T2DM, however, is compromised by several chronic complications. Intensive glucose control, after a follow-up of 15 years, doesn’t produce substantial differences among the groups on cardiovascular outcome dependent on diabetes, it is not associated with significant benefits for the quality of life [1] and it does not prevent T2DM typical chronic complications [2, 3]. In elderly patients there are chronic pain and sensory or motor paraesthesia [4], vegetative neuropathy, peripheral neuropathy, sense organs failure [5], sarcopenia [6] and loss of cognitive functions in relation to cerebral neuropathy [7]. Neurovegetative and somatic complications can coexist [8], interfere with the subjective and affective experience and undermine the relationship between patients and their environment [9, 10].

In T2DM the microvascular disorder is systemic and affects the integrity and the functions of the biological heritage [11]. In elders with T2DM and chronic kidney disease (CKD), without macrovascular precedents, microangiopathy effects the early atrophy of the brain matter of the frontal and temporal lobes and it affects memory, the executive functions and ultimately the behaviour [12]. The addition of vasoactive medications to those antidepressant does not improve mood in patients with depressive syndrome [13-15]. Recent studies have reported interesting clinical effects after administration of negative electric charge (PESF) or after exposure to an electromagnetic field (PEMF) in patients with chronic disease, that cannot be completely attributable to a simple vasodilatation. PESF potentiates microcirculation autonomous motility or “vasomotion” [16], it dissolves or inhibits rouleaux formation [17, 18], increases the baseline of circulating nitric oxide (NO), delivery of oxygen (O2) to the peripheral tissue, basal metabolic rate [19] and, finally, accelerates ulcers healing in patients with T2DM [20]. PEMF dissolves erythrocyte aggregates [21], increases the diameter of microcirculation vessels in T2DM, accelerates blood velocity in cutaneous microcirculation and improves healing of skin wounds [22]. In rat model PEMF stimulates the expression of mRNA HCN1/HCN2 in peripheral nerves with experimental chronic neuropathy [23], limits the outcomes of experimentally induced strokes, acts on neuroinflammation and on circulating NO level [24] and, when administering extremely low-frequency pulses, activates antioxidant defence mechanisms in human osteoblasts [25].

Materials and methods

Patient population

Our study, which was made possible by the generous work of doctors and nurses, was carried out in five years (2009–2014). It involved male and female patients aged over 60 years controlled from glycaemic point of view, in periodic haemodialysis for at least six months. The means to be employed (PESF and questionnaire), the use conditions and the purpose have been fully described to all the candidates. Family members have been involved in the case of dependent patients. In no case the drug intake at home and the rhythm of dialysis were modified. Thirty individuals agreed to participate in the study and were assigned to group A or B with “simple random system”, alternately according to their order of arrival. Group A (mean age 75.70±12.36 years) was composed of 13 women and 12 men (HbA1c = 6.76±0.80%) and group B (mean age 74.93±8.52 years) was composed of 12 women and 13 men (HbA1c = 6.64 ± 0.86%). Group A (treated) was subjected to a real PESF (NewHealtH9000, Akern / RJL Systems) treatment, while group B (control) to a simulated PESF treatment. During the simulated sessions we did not administer negative charges in group B. Each “cycle of PESF”, real and simulated, was performed before dialysis, each session lasted 30 minutes and was repeated three times a week, every other day, for a total of 13 times.

We used impedance tests (BIA-101, Akern / RJL Systems) to establish the intensity of PESF treatment based on each patient’s cell mass index [19] and to monitor the nutritional status of all patients at the end of the PESF cycle.

Blood samples were taken before the start of the dialysis session, at the end of the dialysis and 30 minutes after the end of the purifying session on the first purifying treatment of the week (after the long interdialytic period) at baseline and repeated after the PESF cycle (13 sessions) before starting the dialysis session, at the end and after 30 minutes of dialysis.

The sampling for the electrophoresis control of serum proteins was carried out only before the dialysis session, pre and post PESF.

The results were obtained through observational and retrospective clinical reports using the electronic archive present in our Operating Unit in accordance of STROBE criteria [26]. We excluded patients with major arteries occlusion, valued with doppler test and patients who used: polyphenols, antioxidants for their effects on the cellular signal; phytotherapics (eg. Centella asiatica extracts) for their known effects on endothelial cells; anabolic drugs for their effects on metabolism; and psychotropic drugs.

All participants provided written informed consent and had the opportunity to withdraw from the study without providing any explanation. The protocol was approved by the General Health Director (DS1687_24.02.2010).

Nitric oxide assay

Circulating NO blood levels were tested using ELISA commercial kit (#KGE001, R&D). The method is a spectrophotometric that uses Greiss reaction which is based on enzymatic conversion of nitrates to nitrites due to nitrate reductase [27, 28].

Questionnaire

As the vasodilatation in some psychotic manifestations is unsatisfactory [29], we decided to evaluate, on the basis of questionnaires, the effects on the perceived quality of life in a group of elderly patients before and after exposure to PESF, assuming that the effects on microcirculation, including effects on motility or “vasomotion”, rouleaux breakdown and/or inhibited formation, increased circulating NO level and increased delivery of O2, induce systemic effects which cannot be achieved only by vasodilatation. Individuals with evident microcirculation deficiency, patients with skin wounds, have been chosen for the study. Skin wounds are very common in patients with T2DM and CKD, they depend on the efficiency of microcirculation, and, in the late stages of CKD, they occur more frequently and may affect the quality of life [30].

The questionnaire forms were downloaded from the website [31] and then adapted to our operational reality without changing the purpose and respecting Gordon’s suggestions, who considers the patient a “unique subject” despite its complexity [32]. The monitoring was supposed to ensure changes in self-assessed health in relation to objective limitations imposed by a wide variety of conditions, including perception and management of their own health, food freedom in respect of food patterns, the level of physical activity, cognition and perception capacity, the sleep-wake rhythm and social relationships, which are certainly compromised in case of subjective limitations [33-35]. In addition to the interview the operating model included observation, physical examination, access to the medical records and, in case of doubt, an interview with a family member. The pain that patients had before and after the PESF cycle was assessed by simple scale in which the maximum intensity was 10 and the minimum intensity was 0.

We profiled that an improved tissue perfusion could have effects on artificial blood clearance and on metabolism. To verify the first effects, we checked the azotaemia before, at the end and 30 minutes after the end of the dialysis session (on the first day of dialysis of the week). To determine the second effects, we checked body weight (Kg), phase angle (detected with impedance analysis), azotaemia, blood creatinine, uric acid levels, serum calcium, phosphatemia, hemoglobinemia, haematocrit, globular volume, percentage of albumin (taken from serum protein electrophoresis) and NO, running tests between the dialysis days.

Statistical analysis

Univariable comparisons between groups were performed by χ2 tests or Mann-Whitney U or Spearman rank correlation test. Only 2-tailed p<0.05 was considered statistically significant. The data analysis was generated using SPSS Statistic software, Version 25.0 for IOS. The homogeneity of the groups was checked using Andrew’s curve. This system extracts from the original variables, according linear and hierarchical combinations, the contribution to the total variance [36]. Andrews curves which have been constructed with the first 5 principal components (CP) equal to 92% of the total variance. Principal components: they are extracted, by means of orthogonal rotation, from the original variables according to linearized combinations and according to their contribution to the total variance. Each curve represents a single individual (group A, pre and post PESF; group B, pre and post simulated PESF). Common laboratory tests (azotaemia, creatinine, uricemia, haemoglobin and albumin) were used for each curve.

Results and discussion

The effects on metabolism in group A and B are represented in Fig 1, curve of Andrews [36] where each curve represented a single patient and were generated using common laboratory parameters tests (azotaemia, creatinine, uricemia, haemoglobin and albumin), which are usually used in the clinic in patients with CKD. As can been seen, in group B the dispersal of the curves in quadrant POST is not different from that in quadrant PRE, while in the case of group A, in quadrant POST, a representative bundle of curves shows an evident homogeneity compared to those represented in quadrant PRE. As the original variables, in both groups, relate to the uremic status, it can be considered that PESF could have caused, only in group A, appreciable variations of the metabolic status in nearly all subjects examined. Only in group A the individuals communicate to feel less severe pain and therefore to benefit from the treatment received.

Fig 1

Graphical representation of the PESF effects on some laboratory tests through the Andrews curves which have been constructed with the first 5 principal components (CP) equal to 92% of the total variance.

Each curve represents a single individual (group A, pre and post PESF; group B, pre and post simulated PESF). Common laboratory tests (azotaemia, creatinine, uricemia, haemoglobin and albumin) were used for each curve. The end of the graphs is: along the X axis: -3.14 and 3.14 and along the Y axis: -10 and +10.

In group treated (A) the azotaemia levels, before vs. after the PESF cycle respectively, were pre-dialysis 145.81±24.9 mg/dl vs. 142.56±21.1 mg/dl, p = ns; post-dialysis 48.94±9.4 mg/dl vs. 44.88±4.1 mg/dl, p = 0.09 and after 30 minutes from the end, 58.00±9.7 mg/dl vs. 51.00±4.1 mg/dl, p = 0.013.

In the control group (B) the azotaemia levels, before vs. after the simulated PESF cycle respectively, were pre-dialysis 147.00±21.4 vs. 147.35±21.5 mg/dl, p = ns; post dialysis azotaemia 54.05±12.4 mg/dl vs. 50.00±9.9 mg/dl, p = ns and after 30 minutes, 60.05±11.7 mg/dl vs. 57.45±11.5 mg/dl; p = ns.

The azotaemia level checked 30 minutes after the end of the depurative session is significantly reduced compared to the value measured before PESF cycle, only in group A (Group A pre vs. post, p = 0.01). Moreover, in group A the same value is statistically lower than in the control group B (post: group A vs B, p = 0.02).

The results obtained through the questionnaire in group A and B are shown in Table 1. Indicators in order to document the perceived quality of life before and after PESF suggest that the administration of pulsed negative charges has positive effects only in group A, where the opinion on the physical functions, the limits due to the disease, the limitations due to emotional problems and, in general, on the disease has considerably improved.

Table 1

Results obtained by the health questionnaire SF-31 standard in group A and B.

The results show a significant improvement in the group as regards the physical function, the limits imposed by the disease, emotional problems and overall, the opinion about the disease. Significance was calculated by χ2 tests.

Table 1	Group A			Group B
	PRE	POST	p	PRE	POST	p
Physical functioning	58	74	0.0320	53	45	ns
Role limitation due to physical health	23	79	0.0001	37	38	ns
Role limitation due to emotional problems	38	80	ns	49	44	ns
Energy/Fatigue	53	66	ns	68	60	ns
Emotional well being	59	74	ns	70	70	ns
Social functioning	57	78	ns	68	64	ns
Pain	59	69	ns	74	73	ns
General Health	47	65	0.0136	58	55	ns

In addition, though not explicitly investigated, all have reported that they had improvements in performing common daily activities (household duties and spare time) and that waking hour, resting hours and the perception of the quality of sleep had changed for the better.

Changes in body weight, systolic pressure, diastolic pressure, and heart rate are set out in Fig 2. As can be seen, only in group A, statistically significant variations are recorded. We have illustrated illustrates changes in circulating NO levels in group A, before and after a real PESF treatment, and in group B, before and after a simulated treatment. As can be observed there has been a statistically significant increase of circulating NO level only in group A.

Fig 2

Clinical and laboratory characteristics in Group A e B, pre and post, PESF or not.

Body weight, systolic and diastolic pressure, heart rate changes significantly reduced, while Nitric Oxide increased only in Group A post treatment. Significance was calculated by Mann-Whitney U test.

The impedance test did not show significant changes in the biological heritage in the two groups at the end of treatment for fat, fat-free mass, cell mass, muscle mass and hydration (total water and extracellular). On the contrary, the phase angle is slightly but statistically increased in group A (pre = 4.99±1.2, post 5.48±1.1; p = 0.019) and not changed in group B (pre = 4.97±1.3; post 5.06±1.8; p = ns).

The phase angle expresses the integrity of cell membranes and is therefore indirectly related to cellular activity in Fig 3. The increase in the percentage of albumin in group A alone suggests that PESF may have improved the synthesis or release of albumin, but it is not excluded that PESF has improved vascular permeability and reduced albumin passage in the tissues.

Fig 3

Correlation between baseline levels of phase angle (PA) and albumin (%) in Group A e B, pre and post, PESF or not.

Spearman correlation coefficient and p-value are reported.

Conclusions

In the design phase of the study, we were aware of the extreme difficulty to be able to document neurological clinical benefits in elderly patients with T2DM and CKD after a single PESF cycle for two good reasons: the unavoidable clinical differences between an individual and the other of the same group, and the absence of direct effects of a electrostatic field of low intensity on central neurological tissue [37].

Microcirculation is a set of vessels between the arterial and the venous system and its efficiency is entrusted to the structural and functional integrity of endothelial cell [38]. The evaluation of the microcirculation functions, endothelium-employee, is a common prospective assessment method in the development and progression of chronic cardio-metabolic and/or kidney disease [39].

As we get older microcirculation undergoes significant structural and functional modifications that are the origin of many clinical implications [40]. The process of involution is accelerated in conjunction with chronic disease: vascular network appears rarefied, the vessels are tortuous, the endothelial glycocalyx is deconstructed and the endothelial synthesis of some protein and NO is reduced [39, 41].

The correlation between neuronal cognition and microcirculation dysfunction has been documented in the elderly population without any alteration of macrocirculation’s [42]. Perceived quality of life improvements has been documented by the processing of data collected by a questionnaire before and after the PESF cycle. The unsatisfactory effect of vasoactive medicines, added to antidepressants [15], suggests that vasoconstriction is not presumably the only or the main physical-pathological mechanism. Some people have been reporting for a long time the effects of erythrocyte aggregates or rouleaux on O2 transportation [43].

At present, there are no known molecular targets that relate to the accelerating involution of microcirculation and no medications, that can prevent it, are available. In the elderly population with T2DM and CKD the anti-aging recommendations are like those of other patients and the coexistence of physical and pharmacological fragility [44] are an insurmountable obstacle that cannot be modified even with multidisciplinary approaches [45]. The role of microcirculation is not limited to a passive supply of nutrients [46], but also to an active elimination of uremic toxins [47]. We believe that the extent of accumulation can locally affect the functional integrity and the biological heritage.

Dietary restrictions lead to limited benefits [48] and, when excessively restricted, may favour hypovitaminosis and endothelial dysfunction [49].

Physical limitations negatively affect the intellectual dysfunction and alter the ability of self-management in everyday life and the perceived quality of life [50]. In young chronic renal patients with T2DM vigorous and prolonged physical exercises lead to appreciable effects on physical performance, they lead to self-esteem, with positive effects on anxiety, fatigue and sleep disorder, and ultimately on social reintegration [51]. In elderly patients the possibilities of physical conditioning are limited [52] and early neurocognitive deterioration may encourage self-exclusion and accentuate a sedentary lifestyle and loneliness. It is therefore important to stimulate the physical function in order to minimize the risk of loss of independence and to improve the quality of life in chronic patients. Many have documented these improvements which are attributed to better performance where microcirculation plays an important role [53, 54]. Some have described improvements in athletes undergoing stressful muscle exercises and attributed the results to effects of PESF on microcirculation [55].

Assisting patients with T2DM and CKD is the result of a portentous technological and pharmacological pathway. Clinical results, on the rehabilitation level, are sometimes incredible, and we can’t understand the differences of result even when they are significantly different between a patient and another. Different metabolic conditions could be at the origin of the differences, affect the results and, in chronic patients, affect the perception of the quality of life. We have shown that PESF improves some of the rates of perceived health and makes more homogeneous a group of patients that was previously inhomogeneous as regards the metabolic profile.

The phase angle (PA) is universally considered a sensitive indicator of well-being that varies with changes in health. The reliability of PA in sport requires further investigation but is considered a sensitive indicator of health and integrity of muscle tissue [56, 57], malnutrition [58] and frailty [59]. Our study shows that in elderly hemodialysis patients PESF, after just over a month, did not change the biological heritage (fat mass, lean mass, cell mass and muscle mass) but affected the functional heritage as suggested by the increase in angle of phase.

Patients with CKD have a high incidence of micro and macroangiopathy. The reduced availability of endogenous NO, consequent to endothelial dysfunction, can lead to peripheral vasoconstriction, impaired haemostasis, enhanced platelet activation and above all impaired permeability of the vascular wall [60]. It has been shown that, where there is a certain degree of vascular reactivity, this is often accompanied by changes in electrical charges both on the endothelium and on the surface of the erythrocytes [61]. In patients with chronic kidney disease, hypercholesterolemia (LDL), hyperglycemia, inflammation, over-selling and altered shear stress, can damage and degrade the glycocalyx with loss of negative electrical charges and induce endothelial dysfunction, aggravating the risk of atherosclerosis [62]. From the literature data we therefore hypothesized that the circulating NO level is a good marker of endothelial damage. In addition, vascular compromise is the leading cause of morbidity and mortality in CKD patients [63]. Dialysis techniques do not help depressed endothelial activity [64]. In our opinion, endothelial dysfunction is reversible, and the result affects clinical parameters dependent on vascular resistance [16] and PESF, inducing an increase in NO could affect at least some of the compromised endothelial functions including new vascular resistance with beneficial effects on healing. of difficult wounds in diabetic patients [20]. The use of electric fields of the order of those generated by the bloodstream influences the essential biology of endothelial cells, and the release of NO [65] suggests that the negative electrical charges administered benefited the biosynthetic activity of endothelial NO (eNOS) and presumably erythrocyte (rNOS) NO [65]. Weight reduction and increased levels of NO only in patients who have undergone treatment with PESF confirm its role in the reduction of abdominal obesity [66] in the influences endothelial cell electrophysiology and NO production [65] and the role of NO in weight loss [67]. In this study, we deduced the ameliorative effects of the permeability (selective or not) of the vascular wall from the increase in the phase angle and from the reduction of the controlled blood urea thirty minutes after finishing the dialysis session. To confirm our hypothesis, the impedance test demonstrates that PESF, after only one cycle, by positively modifying PA, acted on the functional and not on the biological patrimony of debilitated people. Hemodialysis sessions of elderly patients on hemodialysis are generally characterized by cardiovascular instability and short interruptions of purification sessions that do not allow to reach the pre-set weight for the end of dialysis (dry weight). The greater weight reduction in group A is connected, as well as with the greater vascular stability, with modified permeability of the endothelium.

Recently some authors have shown that the standardized phase angle, measured with bioelectrical impedance, is a prognostic biomarker independent of age, sex, degree of obesity (BMI) and comorbidity and is unfavorably correlated with mortality in inflamed patients (COVID-19). The patients most at risk are those with alterations of the vascular wall of the peripheral circulation (diabetes, hypertension, dyslipidemia or heart disease) and that the cell mass index and hydration can be identified with PA. Among the biomarkers related to metabolic health they cite albumin [65].

There is no doubt that the degree of “perceived health” affects both individual and social life and it is a necessary condition to achieve an increased family and social wellbeing.

The trained nurse, responsible for nursing, is in direct contact with the patient and can assess the level of satisfaction and the perceived quality of life. The questionnaire is a technical-scientific and management tool that can capture variations related to quality of life, when it is undermined through sickness, and the differences of perceived health. In our case, the nurse listened, informed, involved the patient and, as a close friend, assessed the need for treatment with him before and after the PDF. The self-administered questionnaires allow to measure the transversal differences in terms of quality of life among patients at a given time, within a group of patients or during a given observation period. Even when changes are small but related to individual disease states, groups of patients or functional areas, they can be noticed and reported.

The azotaemia is a waste product that derives from the metabolism of proteins. In patients with impaired renal efficiency, it accumulates first in interstitium and then in blood. The azotaemia also increases during an incongruous protein intake or during an increased protein catabolism. In patients with end-stage chronic renal failure, when the kidneys have become totally insufficient, it is necessary to intervene with replacement therapy to remove waste products. The artificial kidney reduces azotaemia during purification but, when ends dialysis, the catabolites accumulate again until the next dialysis session. Immediately after the end of the dialysis session, the passage of catabolites from the interstitium to the blood causes a new increase in blood urea. Only in group A we have observed statistically lower levels of azotaemia, after 30 minutes from the end of the dialysis session. We believe the result is unexpected, interesting and worthy of further research having observed it in uremic patients characterized by marked protein catabolism. To date, there are no results regarding improvements of azotaemia levels obtained after a short period. We believe that PESF has influenced the metabolic process as evidenced by the increase in albumin and phase angle and is at the origin of less accumulation of nitrogenous products in the interstitium. Our interpretation is supported by the experimental results of other authors who attributed to PESF an increase in the supply of O2 to peripheral tissues and an improvement in basal metabolism in obese individuals [19], greater vascular dynamism (vasomotion) in cardiac patients and accelerated healing of difficult ulcers in diabetic patients [20]. Circulating NO in elderly patients with chronic renal failure is reduced and is correlated with dysfunction of the endothelium and microcirculation. We believe that an increased in circulating NO in group A and although we are unable to specify whether the increase is endothelial (eNOS) or erythrocytic (rNOS), may be expression of an improved vascular performance. The increase in the percentage of albumin, the increase in PA and the positive relationship between albumin and PA are not justifiable if a PESF effect on the endothelial and microcirculation is not accepted. These results agree with those recorded on the best perceived quality of life.

The questionnaire highlighted improvements in physical functioning, on the limits attributed to the role of health (role limitation due to physical health), limitations attributed to the role of emotional problems (role limitations due to emotional problems) and health in general. (General Health).

These results agree with those observed and attributed to the improvement of the phase angle which is a sensitive indicator of health changes. Finally, we believe it is right to specify that we are aware of the effects of biofavonoids and phytotherapeutic substances including the Total Triterpenic Fraction (FTTCA) of Centella asiatica. Both categories of products act on the endothelium and reduce edema. We have previously dealt with arterial ulcers [20]. The derivatives of Centella Asiatica, applied locally, favour the healing of venous ulcers. Centella asiatica improves the supply of O2 to tissues.

We are convinced that the results obtained are attributable to different effects that, although minimal but in a synergistic way, have all contributed to making tissue nutrition more efficient. Moreover, it is clear that “perceived pain” reduction in addition to the increase of physical activity releases from pain, it facilitates interpersonal relations and increases subjective wellbeing. The sense of less dependence reinforced the sense of adequacy of the subject, as documented. Although we cannot rule out that this phenomenon might have been induced by a better tissue perfusion, it is not easily interpreted by mechanisms that exclude tissue perfusion. In our future perspective, it will be necessary to investigate the impact of PESF at the endothelial level through, for example, the assessment of circulating endothelial cells (CEC), a reliable marker of disease activity in a variety of vascular disorders [68] and emerging markers of repair endothelial and activation/apoptosis [69]. The use of the technique in the long term requires further studies.

20 Apr 2021

PONE-D-21-05955

Study of pulsed electrostatic field (PESF) in the perfusion of peripheral tissues: microangiopathy, nutrition and quality of perceiver life.

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Although reviewers found this study interesting, they are concerned about the statistical interpretation of the results. They also found that the authors have made certain assumptions without proper data support. The manuscript at present needs through revision to address the comments and criticisms about technical aspects of the analyses.

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Reviewer #1: The authors present a study on the effect of PESF on elderly patients undergoing hemodialysis. This was a long and involved study that has some interesting observations but for reasons listed below would require significant modification to be accepted for publication.

1. The entire manuscript needs to be checked for grammar and composition. There are significant errors in sentence structure.

2. Are the authors following a standard guideline for clinical research studies - like STROBE - since this appears to be observational? This is unclear.

3. Authors make a lot of assumptions based on very little data that is not clearly laid out. Details below:

a. Figure 1 - very hard to follow - the authors use the words no change (gp B) and homogeneity (gp A) - what is the difference? The figure shows 5 components in Gp B panel and 6 components in Gp A panel. Also note - the word used by authors is componenti - which needs to be corrected. The authors cannot expect a viewer of that figure to know at-a-glance what the difference is pre and post treatment in groups A and B. There must be a clearer representation of the exact differences pointed out or shown in a different format.

b. Authors describe measuring azotaemia levels - but where is the figure to show actual numbers? Only statistics are descriptively included.

b. Although the authors are comparing two cohorts that are age-matched and matched for gender - there is insufficient demographic information to be clear about how other underlying conditions could impact the outcomes of their studies - not discussed at all.

c. It is unclear how soon after the treatment the questionnaire was applied - so is the effect in group A short term or long term?

d. Insufficient details in methodology: What were the simulated and actual test parameters? The authors reference a paper but there needs to be a brief summary of what was applied in this study.

e. The authors point out that NO levels are increased in Group A after treatment. Yet they do not discuss what the significance of this might be? Are the levels physiologically relevant to make an impact?

f. In all other data tables - the pre treatment levels were comparable between group A and B. However in table 3 - group B pretreatment seems quite high (equation (ln) column) compared to the same cohort in group A - can the authors explain that difference?

g. The discussion section is very wordy and reads like a general review - instead of specific and focused comments based on the actual observations they made.

Reviewer #2: Liani et al., studied the effect of of pulsed electrostatic field (PESF) in the perfusion of peripheral tissues: microangiopathy, nutrition and quality of perceiver life. The manuscript has raised some major concerns.

1. In Table 2 the difference in values in pre-and post of group A is not substantial (though the p value is less than 0.05). The SD is large and the number of samples is not that big. Same type of differences are seen in Group B but not significant. The authors must show individual data pints in the form of graph and confirm the p values.

2. The title claims nutrition which is not at all mentioned in the results or discussion

3. Figure 1 is not clear.

Reviewer #3: The study is interesting, was rigorously done for 5 years, the perfusion of peripheral tissues employing employing pulsed electrostatic field was novel, however they were significant gaps, that authors could address these following gaps

1. There are literature evidences suggesting that there is increase in nitric oxide levels & sensitivity with weight loss, authors in the table 2 talk about significant difference in weight loss (p = 0.007), the control group had no weight loss, there is no discussion how the weight loss impacts (given in the table 2), the nitric oxide levels in case of PESF treated and untreated groups, Authors could include few lines in discussion if there is any positive co-relation between weight loss, PESF, and Nitric oxide levels with appropriate references.

2. There is no clear elaboration on why there are no molecular markers included in the study for determining the microangiopathy recovery or progression. Authors could possible include one blood marker to indicate is due recovery due to synergistic effect of PESF other than NO levels.

3. The study would have been more interesting, if the authors could include any other treatment of microangiopathy (example but not limited) total triterpenic fraction of Centella asiatica (TTFCA) as another control group to determine the how positive is PESF bringing the life changes in microangiopathy

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

25 Jun 2021

Dear Reviewers,

thanks for constructive comments which made it possible to improve the paper.

1. The entire manuscript needs to be checked for grammar and composition. There are significant errors in sentence structure.

We have revised grammar composition

2. Are the authors following a standard guideline for clinical research studies - like STROBE - since this appears to be observational? This is unclear.

Our study is an observational and retrospective clinical report using the electronic archive present in our Operating Unit in according of STROBE criteria

3. Authors make a lot of assumptions based on very little data that is not clearly laid out. Details below:

a. Figure 1 - very hard to follow - the authors use the words no change (gp B) and homogeneity (gp A) - what is the difference? The figure shows 5 components in Gp B panel and 6 components in Gp A panel. Also note - the word used by authors is componenti - which needs to be corrected. The authors cannot expect a viewer of that figure to know at-a-glance what the difference is pre and post treatment in groups A and B. There must be a clearer representation of the exact differences pointed out or shown in a different format.

We have clarified Figure 1 and correct word mistake.

b. Authors describe measuring azotaemia levels - but where is the figure to show actual numbers? Only statistics are descriptively included.

We have insert azotaemia numbers

b. Although the authors are comparing two cohorts that are age-matched and matched for gender - there is insufficient demographic information to be clear about how other underlying conditions could impact the outcomes of their studies - not discussed at all.

The two groups were uniform and showed no noteworthy statistical differences

c. It is unclear how soon after the treatment the questionnaire was applied - so is the effect in group A short term or long term?

The effects were evaluated just after the treatment, and it would be interesting to re-evaluate them in the long term

d. Insufficient details in methodology: What were the simulated and actual test parameters? The authors reference a paper but there needs to be a brief summary of what was applied in this study.

We have implemented the methods even though there are references from our previous studies where the treatment details are present

e. The authors point out that NO levels are increased in Group A after treatment. Yet they do not discuss what the significance of this might be? Are the levels physiologically relevant to make an impact?

f. In all other data tables - the pre treatment levels were comparable between group A and B. However in table 3 - group B pretreatment seems quite high (equation (ln) column) compared to the same cohort in group A - can the authors explain that difference?

The pre of group B is not significantly different from group A

g. The discussion section is very wordy and reads like a general review - instead of specific and focused comments based on the actual observations they made.

Thanks we have remodeled the discussion

Reviewer #2: Liani et al., studied the effect of of pulsed electrostatic field (PESF) in the perfusion of peripheral tissues: microangiopathy, nutrition and quality of perceiver life. The manuscript has raised some major concerns.

1. In Table 2 the difference in values in pre-and post of group A is not substantial (though the p value is less than 0.05). The SD is large and the number of samples is not that big. Same type of differences are seen in Group B but not significant. The authors must show individual data pints in the form of graph and confirm the p values.

We confirm the statistics reported

2. The title claims nutrition which is not at all mentioned in the results or discussion

We have insert nutrition profile in discussion

3. Figure 1 is not clear.

We have clarified Figure 1 and correct word mistake.

Reviewer #3: The study is interesting, was rigorously done for 5 years, the perfusion of peripheral tissues employing employing pulsed electrostatic field was novel, however they were significant gaps, that authors could address these following gaps

1. There are literature evidences suggesting that there is increase in nitric oxide levels & sensitivity with weight loss, authors in the table 2 talk about significant difference in weight loss (p = 0.007), the control group had no weight loss, there is no discussion how the weight loss impacts (given in the table 2), the nitric oxide levels in case of PESF treated and untreated groups, Authors could include few lines in discussion if there is any positive co-relation between weight loss, PESF, and Nitric oxide levels with appropriate references.

We have introduced this aspect into the discussion

2. There is no clear elaboration on why there are no molecular markers included in the study for determining the microangiopathy recovery or progression. Authors could possible include one blood marker to indicate is due recovery due to synergistic effect of PESF other than NO levels.

Thanks for the tip we will keep it in consideration for future studies

3. The study would have been more interesting, if the authors could include any other treatment of microangiopathy (example but not limited) total triterpenic fraction of Centella asiatica (TTFCA) as another control group to determine the how positive is PESF bringing the life changes in microangiopathy

We excluded patients with major arteries occlusion, valued with doppler test and patients who used: polyphenols, antioxidants for their effects on the cellular signal; phytotherapics (eg. Centella asiatica extracts) for their known effects on endothelial cells; anabolic drugs for their effects on metabolism; and psychotropic drugs. we believe it is right to specify that we are aware of the effects of biofavonoids and phytotherapeutic substances including the Total Triterpenic Fraction (FTTCA) of Centella asiatica. Both categories of products act on the endothelium and reduce edema. We have previously dealt with arterial ulcers[20]. The derivatives of Centella Asiatica, applied locally, favour the healing of venous ulcers. Centella asiatica improves the supply of O2 to tissues.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

6 Dec 2021

PONE-D-21-05955R1

Study of pulsed electrostatic field (PESF) in the perfusion of peripheral tissues: microangiopathy, nutrition and quality of perceiver life.

PLOS ONE

Dear Dr. Liani,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Jan 20 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

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We look forward to receiving your revised manuscript.

Kind regards,

Kanhaiya Singh, Ph.D

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Additional Editor Comments:

Although reviewers have acknowledge the significant efforts of authors to revise this manuscript, they have recommended some more changes in order to make this study robust for publication. Please include individual data points for conclusions drawn for Table 2. Reviewer 3 comments are mentioned below:

Reviewer 3:

1. There are literature evidences suggesting that there is increase in nitric oxide levels

& sensitivity with weight loss, authors in the table 2 talk about significant difference in

weight loss (p = 0.007), the control group had no weight loss, there is no discussion

how the weight loss impacts (given in the table 2), the nitric oxide levels in case of

PESF treated and untreated groups, Authors could include few lines in discussion if

there is any positive co-relation between weight loss, PESF, and Nitric oxide levels with

appropriate references.

Response: We have introduced this aspect into the discussion

Reviewer 3: Could you please elaborate the discussion further about positive co-relation between weight loss, PESF, and Nitric oxide levels with appropriate references.

2. There is no clear elaboration on why there are no molecular markers included in the

study for determining the microangiopathy recovery or progression. Authors could

possible include one blood marker to indicate is due recovery due to synergistic effect

of PESF other than NO levels.

Response: Thanks for the tip we will keep it in consideration for future studies

Reviewer 3: Could you please add a section on how circulating endothelial cells (CECs) are a reliable marker of disease activity in a variety of vascular disorders. If possible an experiment to determine the increase in count of endothelial cell post PESF.

3. The study would have been more interesting, if the authors could include any other

treatment of microangiopathy (example but not limited) total triterpenic fraction of

Centella asiatica (TTFCA) as another control group to determine the how positive is

PESF bringing the life changes in microangiopathy

Response: We excluded patients with major arteries occlusion, valued with doppler test and

patients who used: polyphenols, antioxidants for their effects on the cellular signal;

phytotherapics (eg. Centella asiatica extracts) for their known effects on endothelial

cells; anabolic drugs for their effects on metabolism; and psychotropic drugs. we

believe it is right to specify that we are aware of the effects of biofavonoids and

phytotherapeutic substances including the Total Triterpenic Fraction (FTTCA) of

Centella asiatica. Both categories of products act on the endothelium and reduce

edema. We have previously dealt with arterial ulcers[20]. The derivatives of Centella

Asiatica, applied locally, favour the healing of venous ulcers. Centella asiatica

improves the supply of O2 to tissues.

Reviewer 3: Could you please include a patient group subjected through a different mode of treatment for microangiopathy as a positive control group

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: (No Response)

Reviewer #3: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

Reviewer #3: (No Response)

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: (No Response)

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: (No Response)

Reviewer #2: Yes

Reviewer #3: (No Response)

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: (No Response)

Reviewer #2: Yes

Reviewer #3: (No Response)

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The reviewers have made a good effort to improve their manuscript. Few points that need addressing in order to accept

1. While the authors did clean up most of their grammatical issues, there are still some that need revising. If you are able to get a professional english editing service to help, that might be very useful. Examples:

a. Patients treated with PESF were improved circulating nitric oxide levels - should read patients treated with PESF 'showed' improved...

b. improper and incomplete internal editing: In elders with T2DM and chronic kidney disease (CKD), without

macrovascular precedents, microangiopathy effects the early atrophy of the brain matter of the frontal

and temporal lobes and it affects memory, the executive functions and ultimately the behaviourFare

clic o toccare qui per immettere il testo.[12].

c. present in our Operating Unit in according of STROBE criteria - should read - present in our operating unit in "accordance with or following" STROBE criteria.

2. No response given to: e. The authors point out that NO levels are increased in Group A after treatment. Yet

they do not discuss what the significance of this might be? Are the levels

physiologically relevant to make an impact?

Perhaps the authors did make edits to account for this but it is not immediately clear.

3. I don't see figure legends included for the 2 figures.

4. Table cannot be shortened to Tab. Please write out the word Table.

I think with these edits, the manuscript may be ok to be accepted for publication.

Reviewer #2: In Table 2 the difference in values in pre-and post of group A is not substantial

(though the p value is less than 0.05). The SD is large and the number of samples is

not that big. Same type of differences are seen in Group B but not significant. The

authors MUST SHOW individual data pints in the form of graph and confirm the p values.

Reviewer #3: (No Response)

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

8 Feb 2022

Date: Dec 06 2021 07:59AM

To: "Rossella Liani" rossellaliani@yahoo.it

From: "PLOS ONE" plosone@plos.org

Subject: PLOS ONE Decision: Revision required [PONE-D-21-05955R1]

PONE-D-21-05955R1

Study of pulsed electrostatic field (PESF) in the perfusion of peripheral tissues: microangiopathy, nutrition and quality of perceiver life.

PLOS ONE

Additional Editor Comments:

Although reviewers have acknowledge the significant efforts of authors to revise this manuscript, they have recommended some more changes in order to make this study robust for publication. Please include individual data points for conclusions drawn for Table 2. Reviewer 3 comments are mentioned below:

Reviewer 3:

1. There are literature evidences suggesting that there is increase in nitric oxide levels

& sensitivity with weight loss, authors in the table 2 talk about significant difference in

weight loss (p = 0.007), the control group had no weight loss, there is no discussion

how the weight loss impacts (given in the table 2), the nitric oxide levels in case of

PESF treated and untreated groups, Authors could include few lines in discussion if

there is any positive co-relation between weight loss, PESF, and Nitric oxide levels with

appropriate references.

Response: We have introduced this aspect into the discussion

Reviewer 3: Could you please elaborate the discussion further about positive co-relation between weight loss, PESF, and Nitric oxide levels with appropriate references.

Response: We have introduced this aspect into the discussion

2. There is no clear elaboration on why there are no molecular markers included in the

study for determining the microangiopathy recovery or progression. Authors could

possible include one blood marker to indicate is due recovery due to synergistic effect

of PESF other than NO levels.

Response: Thanks for the tip we will keep it in consideration for future studies

Reviewer 3: Could you please add a section on how circulating endothelial cells (CECs) are a reliable marker of disease activity in a variety of vascular disorders. If possible an experiment to determine the increase in count of endothelial cell post PESF.

Response: We have introduced this aspect into the discussion but the study is over and the CEC are valued on fresh sample (DOI: 10.1016/j.jim.2012.03.007; DOI: 10.1038/s41598-021-88941-x)

3. The study would have been more interesting, if the authors could include any other

treatment of microangiopathy (example but not limited) total triterpenic fraction of

Centella asiatica (TTFCA) as another control group to determine the how positive is

PESF bringing the life changes in microangiopathy

Response: We excluded patients with major arteries occlusion, valued with doppler test and

patients who used: polyphenols, antioxidants for their effects on the cellular signal;

phytotherapics (eg. Centella asiatica extracts) for their known effects on endothelial

cells; anabolic drugs for their effects on metabolism; and psychotropic drugs. we

believe it is right to specify that we are aware of the effects of biofavonoids and

phytotherapeutic substances including the Total Triterpenic Fraction (FTTCA) of

Centella asiatica. Both categories of products act on the endothelium and reduce

edema. We have previously dealt with arterial ulcers[20]. The derivatives of Centella

Asiatica, applied locally, favour the healing of venous ulcers. Centella asiatica

improves the supply of O2 to tissues.

Reviewer 3: Could you please include a patient group subjected through a different mode of treatment for microangiopathy as a positive control group

Response: Thanks for the tip we will keep it in consideration for future studies, but the study is over

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: (No Response)

Reviewer #3: (No Response)

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

Reviewer #3: (No Response)

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: (No Response)

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: (No Response)

Reviewer #2: Yes

Reviewer #3: (No Response)

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: (No Response)

Reviewer #2: Yes

Reviewer #3: (No Response)

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The reviewers have made a good effort to improve their manuscript. Few points that need addressing in order to accept

1. While the authors did clean up most of their grammatical issues, there are still some that need revising. If you are able to get a professional english editing service to help, that might be very useful. Examples:

a. Patients treated with PESF were improved circulating nitric oxide levels - should read patients treated with PESF 'showed' improved...

b. improper and incomplete internal editing: In elders with T2DM and chronic kidney disease (CKD), without

macrovascular precedents, microangiopathy effects the early atrophy of the brain matter of the frontal

and temporal lobes and it affects memory, the executive functions and ultimately the behaviourFare

clic o toccare qui per immettere il testo.[12].

c. present in our Operating Unit in according of STROBE criteria - should read - present in our operating unit in "accordance with or following" STROBE criteria.

Thanks for the suggestions, we have corrected in the text

2. No response given to: e. The authors point out that NO levels are increased in Group A after treatment. Yet

they do not discuss what the significance of this might be? Are the levels

physiologically relevant to make an impact?

Perhaps the authors did make edits to account for this but it is not immediately clear.

Response: We have improved this aspect into the discussion

3. I don't see figure legends included for the 2 figures.

Response: Thanks. We have included figure legends for the 2 figures

4. Table cannot be shortened to Tab. Please write out the word Table.

Response: Thanks.

I think with these edits, the manuscript may be ok to be accepted for publication.

Reviewer #2: In Table 2 the difference in values in pre-and post of group A is not substantial

(though the p value is less than 0.05). The SD is large and the number of samples is

not that big. Same type of differences are seen in Group B but not significant. The

authors MUST SHOW individual data pints in the form of graph and confirm the p values.

Response: Thanks, but the p value is at most equal to 0.021 in Table 2, and not near to 0.05, full significance P value

Reviewer #3: (No Response)

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Submitted filename: Response to Reviewers_2.docx

Click here for additional data file.

28 Feb 2022

30 Mar 2022

Reviewer #2: I have repeatedly been asking for the individual data points in Table 2. The authors MUST SHOW individual data pints in the form of graph or provide the excel sheet for us to review. THIS HASN'T BEEN ADDRESSED.

Dear Reviewer #2: we have replaced table 2 with the new figure 2 (dot plot) where the single points are represented and repeated the statistic using the most appropriate Mann Whitney non-parametric test.

Submitted filename: Response to Reviewers_3.docx

Click here for additional data file.

3 May 2022

Study of pulsed electrostatic field (PESF) in the perfusion of peripheral tissues: microangiopathy, nutrition and quality of perceiver life

PONE-D-21-05955R3

Dear Dr. Liani,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Kanhaiya Singh, Ph.D

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

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Reviewer #2: Yes

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Reviewer #2: Yes

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Reviewer #2: All comments addressed satisfactorily. This manuscript is now ready to be accepted in its present form.

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Reviewer #2: No

10 May 2022

PONE-D-21-05955R3

Study of pulsed electrostatic field (PESF) in the perfusion of peripheral tissues: microangiopathy, nutrition and quality of perceiver life.

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