The surgeon was not always the same, but all data was collected by one of the authors, J.N., which is now mentioned.

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1 Author's response to reviews Title: Minimal Shedding of the Glycocalyx Layer During Abdominal Hysterectomy. A Preliminary Report Authors: Robert G Hahn (r.hahn@telia.com) Janis Nemme (janis.nemme@gmail.com) Camilla Krizhanovskii (camilla.krizhanovskii@sll.se) Stelia Ntika (stelia.ntika@sll.se) Olegs Sobelnikov (olegs.sabelnikovs@rsu.lv) Indulis Vanags (Indulis.Vanags@rsu.lv) Version: 1 Date: 16 Jun 2017 Author s response to reviews: - It is commendable that the authors use human subjects for the experiment. The authors should make mention of the institutional approval obtained for studying human subjects, in the body of the methods section of the manuscript (the approval is first mentioned in the disclosure section, which is late in the manuscript). Seven human subjects participated in the experimental group. There is a sense that the sample size should be larger, to help definitively prove the impact of hypervolemia on glycocalyx shedding. A power analysis for sample size determination is missing, but it could help confirm that seven subjects is sufficient or it could clarify that more are needed. Did the same physician perform all of the surgeries to reduce human error? AUTHOR S REPLY The information about ethics approval is now given also in the beginning of the Methods section. The surgeon was not always the same, but all data was collected by one of the authors, J.N., which is now mentioned. Two power analyses are now mentioned. The first one is the pre-study analysis, which is given at the end of the Methods section and runs as follows: "The planned clinical trial will finally include 24 patients, which is based on a difference in the rise of the plasma concentration of syndecan-1 of 50% between propofol and sevoflurane anaesthesia (power 80% and P< 0.05)."

2 The second is a post hoc analysis reported in the Results section, which runs as follows: "A post hoc power analysis showed that 120 patients would be needed to demonstrate statistical significance by P< 0.05 for the minimal increase in syndecan-1, which was the key outcome measure, that occurred between the pre- and the postoperative sampling points." - The use of human subjects presents challenges for performing a well controlled experiment with sufficient sample size. To reinforce the statistical significance (or non significance) of the study and to make the results more compelling, a relevant animal model should be used. AUTHOR S REPLY It is true that an animal model could have been an alternative, but based on previous work on volume loading in humans (see Rehm 2001, ref.12) we indeed believed that volume loading of the present magnitude would cause shedding and marked fluid shifts. However, already our first 7 patients strongly suggest that these phenomena are unlikely to occur with the crystalloid fluid volumes used in clinical practice. - The methods are generally well described. However, many specific details are lacking, giving the impression that the study is not complete. (1) It is not completely clear what various forms of general anesthesia were used. Does this study use one drug or two or three - midazolam, fentanyl, and/or propofol - consistently through the study? What specific outcomes come from the use of each form of anesthesia? (2) What is the basis for selecting the glycocalyx components that are measured here, and for ignoring other components? (3) The protocols for identifying and measuring shed glycocalyx components were vaguely described. Also, the authors appear to be measuring glycocalyx shed from the vascular wall as well as glycocalyx shed from the gastrointestinal wall. However, the introductory comments of the paper suggest that these vascular (plasma) and gastrointestinal (urine) measurements are not well linked to the investigative rationale, objectives, and hypothesis. Please elaborate. (4) The authors never mentioned the use of a control group. There should be one control group of patients who did not undergo surgery. There should be another control group in which Ringer's lactate was not administered (or lower in amount) for the early stages of the surgery, because the lack of (or reduced) hypervolemia may in fact help to clarify the effect that hypervolemia has on glycocalyx shedding. (5) It would be interesting to assess whether glycocalyx shedding is more enhanced near the surgery site or near the catheter insertion site, in comparison to systemic levels of glycocalyx shedding. (6) Statistics are appropriate for the distribution and sample size. The nonparametric (median) statistics is less commonly used, so it is suggested that the authors elaborate on the description of this statistics. AUTHOR S REPLY

3 All patients received the same drugs for induction of anaesthesia as reported, but the maintenance differed and could either comprise either propofol or sevoflurane. With such a small group of patients there is no meaning of reporting results separately for these two groups here, but I can say here that they seem to behave very similarly. The shedding products analysed here are commonplace for this type of study, and agrees well with what is reported by others. We can mention that it is very rare that a study contains data on all three components, and that urinary concentrations are reported as well should be regarded as an asset. This is quite rare, too. Despite reading much literature on this topic, we are not aware of the shedding products differ between the blood vessels in the gastrointestinal wall and elsewhere. With regard to the urinary concentrations these are sometimes reported, but no comparison between plasma and urine has been published so far. Therefore we think that reporting both plasma and urine concentrations of these shedding products are of interest to the medical community. No study of concentrations of shedding products near and distant to the surgical site has been performed. Such an approach would require the placement of invasive catheters in the body, which will need to be motivated well for any ethics committee. The statistics now explained better. A control group, with no intervention, would most likely show no change at all in any of the biomarkers, and more reflect the baseline variability. In any event, a control group was not included. - What is the specific efficiency of filtration by the kidneys? Could this explain why measurements were taken for up to 90 minutes post-surgery? Won't this value change when glycocalyx is shed? The authors should include this information in the paper. AUTHOR S REPLY As far as we can see, nothing is previously known about the relationship between plasma and urinary concentrations of these shedding products. No specific measurements of the filtration efficiency were made. - The results section and figure captions need more detail in the text, to create more meaning for the reader. Also, the use of only two figures suggests that this technical report may be preliminary in scope unless the authors are publishing a short communication.

4 AUTHOR S REPLY--- Some more information has been added to the Results section and to the Legends for figures. Yes, this is a preliminary report, that is true. - In the discussion, the reported data was poorly addressed and interpreted. Much of the discussion focused on reviewing previously published literature, although focusing on strong data interpretation and supportive arguments would have made the conclusion more compelling. For example, it would be interesting to elaborate upon why heparan sulfate is so stable in the period immediately following surgery, but not before infusion of fluid and not in the long-term postoperative period. In addition, what is the significance of similar heparan sulfate levels in both plasma and urine? Also, explain what the delayed (60 minutes post-surgery) increase in hyaluronic acid indicates. What does the difference in plasma and urine hyaluronic acid levels indicate? In the first paragraph of the discussion the authors mention interest in albumin leakage from capillaries, but this was never measured. The authors could have easily conducted an albumin leakage experiment using an animal experiment run in parallel. - Lastly, more discussion of the long term goals and implications of this work is highly suggested. AUTHOR S REPLY--- More information has been added to the Results section and to the Legends for figures. The new new passages highlighted in red colour. The late increase in heparan sulfate concentration is likely to be due to surgery-induced inflammation (rather than to ANP-induced shedding). We now say so. Moreover, we interpret the virtually identical concentrations of heparan sulfate in plasma and urine to indicate that this glycosaminoglycan readily equilibrates between is body fluids. The long term goal and implications are now given at the end of the Discussion section as follows: "The long term goal of the current project study is to quantify capillary leakage of albumin and crystalloid fluid kinetics in relationship to shedding of the glycocalyx, as well as to examine if the degree of shedding differs depending on whether propofol (intravenous) and sevoflurane (gaseous) anaesthesia is used. However, the minimal evidence of shedding found in the first 7 patients makes it unlikely that shedding will later be found to markedly influence capillary leakage and fluid kinetics." _ Elaheh Rahbar (Reviewer 2): This is a small pilot study where 7 patients undergoing abdominal surgery were serially sampled for urine and plasma samples. These biological specimens were used to quantify the amount of glycocalyx shedding during the surgical procedure. While the

5 concept of glycocalyx shedding is important, there are several concerns that should be addressed prior to publication. Major Concerns: 1. One of the major concerns from this paper, is that the authors claim no significant glycocalyx shedding based on the syndecan-1 data, despite the fact that there were changes in hyaluronic acid and heparan sulfate concentrations. While syndecan-1 is considered to be one of the main components of the EGL, the other 2 proteoglycans measured are important as well, particularly from an immunomodulatory and anti-coagulant standpoint. Secondly, the authors state that there is evidence of ~37% hemodilution, but do not use this to adjust the measured EGL concentrations. My understanding is that if syndecan-1 concentrations did not change much in the presence of ~37% hemodilution, is that you may actually have higher shed amounts of syndecan-1 (by mass) but they do not translate to higher concentrations since the plasma volume may have potentially increased due to infusion of 2L of LR. AUTHOR S REPLY--- We claim that changes were minimal so as to greatly differ from the quadrupled changes in the concentrations of shedding products reported elsewhere (see numerous citations in our paper). It is unlikely that the small changes reported here will change the permeability of the endothelium for macromolecules in any important way. "In the present study we recorded a marked plasma volume expansion at the end of infusion, while distribution throughout the extracellular fluid space is known to reduce crystalloid-induced hypervolemia to only about 15-20% of the infused volume 30 min later [16]. However, no dip in the plasma concentrations of any of the shedding products can be discerned at the end of the infusion as compared to the adjacent data points, which is consistent with that syndecan-1 diffused quickly from the interstitial fluid to the plasma as soon the latter was diluted with crystalloid fluid (Fig. 1, top row). Similar equilibration of substances across the capillary wall on infusion of fluid has previously been reported for other plasma substances, such as cystatin C [17]." 2. Another main concern is the impact of anesthesia on the measured analytes. Did all 7 patients receive the same total amount of anesthesisa? If not, these volumes should be reported and adjusted for, perhaps in Table 1. It is likely that serum heparan sulfate levels could be influenced by the anesthesia received. AUTHOR S REPLY. The following passage is not added under the Limitations section in the Discussion: "The same drugs and doses were used during the induction of anaesthesia, but

6 maintenance was achieved with either propofol of sevoflurane between which a difference in shedding products cannot be evaluated in this preliminary report." 3. The authors try to calculate the change in plasma volume by using hemoglobin and hematocrit values. While I understand that this is a surrogate measure, it is hard to know if the plasma volume indeed changed over this short acute period. I would recommend using the term 'hemodilution' instead of % plasma volume expansion. AUTHOR S REPLY. Haemodilution is now used as a term. However, one should note that we do not report is not haemodilution as such, but a figure after conversion to plasma volume expansion by dividing the change in blood haemoglobin by (1- haematocrit). The use of haemoglobin dilution to indicate changes in plasma volume is quite common, and can be seen in ref 14, 16, 17, 18, 21 and 22. However, the degree of plasma volume expansion is not essential here, but the fact that as much as 25 ml/kg of Ringer s solution was infused over 30 min without causing marked shedding of the glycocalyx. Clinicians can convert this volume to what he used in his own patients, and be confident that crystalloid fluid in normal volumes does not shed the glycocalyx. This is the point where there has been uncertainty. 4. Regarding statistics, when you have multiple time points, I would have recommended a repeated measures ANOVA or longitudinal GEE model to evaluate changes over time. Perhaps some statistical consulting could improve the data analysis. AUTHOR S REPLY. Repeated-measues ANOVA is difficult to apply because data were usually not normally distributed. To make the presentation simple, we summarized data over a reasonable period of time and compared data before surgery with data after surgery and compared these periods with non-parametric statistics. 5. In the results, authors state that no other fluids were given. Does this include blood products? AUTHOR S REPLY. Yes, this also includes blood products. This is now mentioned.

7 6. How was blood loss calculated? Is this controlled bleeding from the surgery? Further, from the data it appears that the blood loss is minimal, so is LR solely provided because of delivery of the anesthesia and to maintain fluid balance (but not because you need to increase plasma volume). This leads to my next concern. AUTHOR S REPLY. Blood loss was quite small, that is correct, and this is normally estimated visually from blood on swabs and dressings, and as measured in suction bottles. This is now mentioned. Fluid during surgery is needed, even without blood loss, to compensate for drug-induced vasodilatation and the re-distribution of blood flow that occurs with it. In particular, blood flow is directed away from the gastrointestinal tract where local hypoxia might develop. Fluid will also accumulate in the cavity created by the removal of the uterus. 7. Rationale for why the glycocalyx may be particularly important in the context of abdominal hysterectomy is not provided. Does this surgery often use a lot of crystalloid fluid infusions? Or are there several inflammatory complications after surgery that could be remediated during surgery? Some extra rationale supporting the author's idea should be added in the introduction. AUTHOR S REPLY. The rationale for using abdominal hysterectomy for the study is now presented in the Introduction. Fluid in a volume of 2 L is commonly infused during abdominal hysterectomy, although the brisk volume loading given at the beginning of the surgery is not typically given. This is now mentioned. 8. Chondroitin sulfate is a major component of the glycocalyx and should be included in the introduction. AUTHOR S REPLY. Done. 9. Not sure how the glycocalyx counteracts oxidative stress. Could the authors provide more detail about this? AUTHOR S REPLY. This notion has been deleted.

8 10. Authors state that hypervolemia can cause glycocalyx shedding, but I have not found enough scientific evidence to suggest this. Rather hypervolemia could exacerbate shedding, but I have not found evidence to support that it is the initial causing factor. AUTHOR S REPLY. Shedding on the endothelial glycocalyx layer occurs in response to release of atrial natriuretric peptides (ANPs) which is demonstrated in Ref. 11 and it is well known that the ANP level is increased by hypervolaemia (see Ref. 19 and others). Marked fluid shifts, probably due to shedding, are reported in hypervolaemia patients given in Ref 12 and commented upon in ref 13. Avoidance of hypervolaemia due to shedding is a commonly given clinical recommendation, see Ref First sentence of discussion, authors claim that degradation of the glycocalyx is an adverse effect of anesthesia. I am not familiar with this. Can authors provide a reference to support this statement? AUTHOR S REPLY. This statement is removed.