Correlation of optic nerve and optic nerve sheath diameter with intracranial pressure in pigs.

OBJECTIVE: Several studies have shown an association between intracranial pressure and the diameter of the optic nerve sheath measured by transbulbar ultrasonography. To understand the pathophysiology of this phenomenon, we aimed to measure the changes of the optic nerve, optic nerve sheath and perineural space separately with increasing intracranial pressure in a porcine model.
METHODS: An external ventricular drain was placed into the third ventricle through a right paramedian burrhole in eight anesthesized pigs. The diameters of the optic nerve and the optic nerve sheath were measured while the intracranial pressure (ICP) was increased in steps of 10mmHg from baseline up to 60 mmHg.
RESULTS: The median diameters of the optic nerve (ON) increased from 0.36 cm (baseline- 95% confidence interval (CI) 0.33 cm to 0.45 cm) to 0.68 cm (95% CI 0.57 cm to 0.82 cm) at ICP of 60 mmHg (p<0.0001) and optic nerve sheath (ONS) from 0.88 cm (95% CI 0.79 cm to 0.98 cm) to 1.24 cm (95% CI 1.02 cm to 1.38 cm) (p< 0.002) while the median diameter of the perineural space (PNS) (baseline diameter 95% CI 0.40 cm to 0.59 cm to diameters at ICP 60 95% CI 0.38 cm to 0.62 cm) did not change significantly (p = 0.399). Multiple comparisons allowed differentiation between baseline and values ≥40 mmHg for ON (p = 0.017) and between baseline and values ≥ 50mmHg for ONS (p = 0.006). A linear correlation between ON (R2 = 0.513, p<0.0001) and ONS (R2 = 0.364, p<0.0001) with ICP was found. The median coefficient of variation for intra- and inter-investigator variability was 8% respectively 2.3%.
CONCLUSION: Unexpectedly, the increase in ONS diameter with increasing ICP is exclusively related to the increase of the diameter of the ON. Further studies should explore the reasons for this behaviour.

Introduction

To obtain accurate transbulbar ultrasonographic measurements the investigator must be familiar with the anatomical structures inside the orbit. Both the optic nerve (ON) and the optic nerve sheath (ONS) appear as hypoechogenic structures. The ON is separated from the ONS by the perineural space (PNS) which is a hyperechogenic layer surrounding the ON (Fig 1) [1].

Fig 1

The upper transbulbar ultrasound image was recorded at baseline ICP–the lower image was recorded at ICP of 40mmHg.

All measurements were obtained 3 mm behind the papilla as indicated by the two stars connected with the dotted line. Optic nerve diameter increased from 0.36 cm to 0.48 cm as illustrated by the black arrow. Optic nerve sheath diameter increased from 0.79 cm to 0.86 cm as illustrated by the white arrow. In both ultrasound images the lens was bypassed in order to obtain the most accurate display of the posterior structures of the eye. Further both images are made by using a zoom box of 2 cm size.

Several studies have shown that the diameter of the optic nerve sheath (ONS) correlates with changes in intracranial pressure (ICP) [2-4]. In critically ill patients, relative but not absolute ICP changes can be monitored bedside using ultrasound [5-7]. Hansen et al. studied ONS changes in patients undergoing lumbar infusion tests when cerebrospinal fluid (CSF) pressure was gradually raised. This was associated with a linear increase in ONS [8]. In a porcine model, a balloon catheter in the superior vena cava led to elevated cephalic venous pressure with consecutive ICP increase which could be tracked by increasing ONS diameter [9]. Further dynamic ONS assessment has been used in the diagnosis of spontaneous intracranial hypotension by determining the respective change between supine and upright body position [10].

So far, studies have not differentiated changes in diameter of ON and PNS with increasing ICP. Since PNS is a subarachnoid cistern, one would assume that acute ICP changes expand this CSF filled space. On the other hand, capillaries and venules within ON may become congested with increasing ICP and ON diameter may therefore increase as well. The aim of our study is to characterize the individual behaviour of ON and PNS with increasing ICP levels. We hypothesized that both ON and PNS diameter increase with increasing ICP.

Further we aimed to assess the accuracy and the reproducibility of transbulbar ultrasonography by measuring the intra- and inter-investigator variability.

Materials and methods

The study was performed in accordance with the Swiss national guidelines for the Care and Use of Laboratory Animals, National Academy of Sciences (1996). An approval of the Commission of Animal Experimentation of the Canton Bern, Switzerland was obtained (approval number BE 40/16). This manuscript adheres to the applicable ARRIVE guidelines.

In compliance with the three R principle, the same group of pigs were subject to three different, unrelated studies, all involving ultrasound. First, a study investigating muscle compartment pressure in both lower legs was conducted [11]. After a stabilisation phase and establishment of a second baseline, a study on abdominal pressure was obtained [12]. Finally, this present study was performed.

Animal preparation

Pigs were chosen because of their size which allowed instrumentation similar to that in humans. Animal preparation was described in detail previously [11-13]. In brief, 8 domestic pigs (weight 39±2kg (mean±SD)) were anesthetized using 20mg/kg ketamine and 2mg/kg xylazine intramuscularly. Midazolam (0.5mg/kg) and atropine (0.02mg/kg) were administrated and the animals were orally intubated. An intravascular catheter was placed in the left internal carotid artery in order to invasively monitor blood pressure.

At the end of the experiment a bolus of 40 mmol potassium chloride was given to sacrifice the animal.

Hemodynamic monitoring

Pulse oximetry and arterial pressure (MAP) were recorded with pressure transducers (xtrans®, Codan Medical, Germany). The recorded values were displayed continuously on a multi-modular monitor (S/5 Critical Care Monitor®; Datex-Ohmeda, GE Healthcare, Helsinki, Finland).

External ventricular drain placement

The pigs were positioned in a left lateral decubitus position for all subsequent study interventions and measurements. A right-sided external ventricular drain catheter with a bolt-kit system for fixation and an air-pouch balloon situated at the tip for ICP measurement (Silverline-Bolt-Kit-catheter®; Spiegelberg GmbH&Co.KG; Hamburg, Germany) was placed in the third ventricle as described elsewhere [14]. In brief, a 7cm midline skin incision over the frontal and parietal bones was made and the sagittal and coronary sutures were exposed. A manually operated twist drill was used to place the burr hole 1cm lateral to the sagittal suture at the upper part of the frontal bone. The dura was perforated and the bolt was fixed with its trajectory aiming towards the midline at an angle of 90° against the frontal bone. Then, the EVD catheter was carefully inserted until CSF could be extracted. Insertion depth was typically 6.5 to 7 cm. For continuous ICP measurement an ICP monitor (Spiegelberg GmbH&Co.KG; Hamburg, Germany) was used. The ventricular drain was connected to an external drainage and monitoring system (Duet External Drainage and Monitoring System®, Medtronic Inc., Minneapolis, Minnesota, USA). In order to ascertain stable ICP levels the EVD catheter was connected to a surge chamber filled with normal saline. By adjusting the height of this surge chamber the respective ICP levels were obtained.

Ultrasound measurements

Ultrasound examinations were performed using a Philips iU22 ultrasound machine (Philips healthcare, Andover, MA, USA) with a 7-17MHz linear array transducer (L15-7io). The mechanical index was set at 0.3 [14-16]. Ultrasound loops were recorded at baseline and 10 minutes after achieving stable ICP target values of 20, 30, 40, 50 and 60 mm Hg. At each ICP step, three consecutive ultrasound examinations were conducted. Transbulbar sonography was only performed on the right side since all pigs were positioned in a left lateral decubitus position. All ultrasound loops were analysed offline by two investigators who were blinded to the intracranial pressure level and to the animal. As per convention, the diameters of interest (optic nerve and optic nerve sheath) were measured 3 mm behind the papilla [2, 3]. The average of the means of the three consecutive measurements from each investigator was used. The diameter of the perineural space (PNS) was calculated by subtracting the diameters of ON from ONS.

The areas of ON and ONS at baseline and at ICP values of 60 mmHg were calculated using the formula area = π / diameter 2. The area of the perineural space was calculated by subtracting the optic nerve area from the optic nerve sheath area.

Statistical analysis

Despite normal data distribution (Shapiro-Wilk-Test), values are displayed as median, range and 95% confidence intervals for better interpretation of data distribution. The effect of increasing intracranial pressure on the different diameters was assessed using Friedman Test. In order to determine at which ICP the diameter became significantly different compared to baseline, each consecutive ICP value was compared to baseline using Dunn’s test for multiple comparisons. Regression analysis were used for identifying the relationship between intracranial pressure and ultrasound assessments. Intra- and inter-investigator comparisons were made using coefficients of variation. ANOVA for repeated measurements with two within-group factors (ICP level and investigator) was used to assess a possible interaction of investigator and diameter with increasing ICP levels. Standard statistical software package was used for analysis of data (GraphPad Prism 8, GraphPad Software, USA).

Results

Placement of the external ventricular drain catheter was successful in all eight animals. Median baseline ICP was 8 mmHg (range 6 to 12 mmHg). Hemodynamic characteristics for each ICP level are presented in Table 1. The sonographic measurements could be obtained for every ICP level in all animals. The inter-investigator coefficient of variation was 2.3% (range 0% - 9.4%). The intra-investigator coefficient of variation was 8% (range 0% - 21%). Details are presented in Table 2.

Table 1

Cardiopulmonary characteristics.

Stage ICP	Baseline	20	30	40	50	60	Friedmann Test
MAP (mmHg)	82 [79–108]	82 [71–105]	89 [72–122]	105 [76–158]	110 [79–179]	156 [79–203]	p = 0.006
Heart rate (beats/min)	88 [76–123]	101 [94–132]	97 [78–155]	126 [81–156]	118 [79–206]	137 [83–250]	p = 0.172
CPP (mmHg)	75 [72–103]	62 [51–85]	59 [42–92]	65 [36–118]	60 [29–129]	98 [19–143]	p = 0.255
pCO2 (mmHg)	39 [35–41]	38 [34–41]	38 [37–39]	38 [36–40]	38 [37–40]	40 [38–44]	p = 0.258

MAP = mean arterial pressure. CPP = cerebral perfusion pressure. Values are given as median and range.

Table 2

Inter- and intra-investigator coefficients of variation.

	optic nerve diameter	optic nerve sheath diameter	all structures
Inter-investigator coefficient of variation overall	2.7% [0.3–9.4%]	2.2% [0–8.6%]	2.3% [0–9.4%]
Baseline ICP level	3.0% [0.3–7.7%]	1.5% [0–6%]	2.2% [0–7.7%]
ICP level 20mmHg	4.5% [0.9–9.4%]	2.9% [1.3–5.7%]	4.0% [0.9–9.4%]
ICP level 30mmHg	2.4% [1.3–8.1%]	1.9% [0.1–5.2%]	2.1% [0.1–8.1%]
ICP level 40mmHg	3.6% [1.3–6.3%]	7.7% [0.3–8.6%]	3.8% [0.3–8.6%]
ICP level 50mmHg	1.8% [1.1–7.8%]	2.8% [0.1–8.3%]	2.1% [0.1–8.3%]
ICP level 60mmHg	1.3% [0.3–6.3%]	2.4% [0.2–7.7%]	1.7% [0.2–7.7%]
Intra-investigator coefficient of variation overall	10% [1–21%]	5% [0–20%]	8% [0–21%]
Baseline ICP level	8.5% [3–21%]	7.5% [0–15%]	7.5% [0–21%]
ICP level 20mmHg	10.5% [2–18%]	5.5% [2–14%]	7.0% [2–18%]
ICP level 30mmHg	9.5% [1–17%]	4.0% [1–14%]	7.5% [1–17%]
ICP level 40mmHg	7.5% [3–16%]	3.5% [1–18%]	5.0% [1–18%]
ICP level 50mmHg	7.0% [1–17%]	5.0% [0–15%]	7.0% [0–17%]
ICP level 60mmHg	5.5% [0–21%]	6.0% [1–20%]	5.5% [0–21%]

ICP = intracranial pressure. All structures = optic nerve diameter and optic nerve sheath diameter. Values are given as median and [range]

The diameters of the ON (baseline diameters 95% CI 0.33 cm to 0.45 cm to diameters at ICP 60 mmHg 95% CI 0.57 cm to 0.82 cm) and ONS (baseline diameters 95% CI 0.79 cm to 0.98 cm to diameters at ICP 60mmHg 95% CI 1.02cm to 1.38cm) increased significantly with increasing ICP level (p <0.002, full range of values Fig 2). However the diameter of the perineural space (baseline diameter 95% CI 0.40 cm to 0.59 cm to diameters at ICP 60 95% CI 0.38 cm to 0.62 cm) did not change significantly (p = 0.399). Multiple comparisons allowed differentiation between baseline (95% CI 0.34 cm to 0.45 cm) and values ≥40 mmHg for ON (95% CI 0.51cm to 0.66cm; p = 0.017) and between baseline (95% CI 0.79 cm to 0.98 cm) and values ≥50 mmHg for ONS (95% CI 1.02 cm to 1.34 cm; p = 0.006). The increase of ICP was paralleled by a linear increase of diameters of both ON (R2 = 0.513, p<0.0001) and ONS (p R2 = 0.364, <0.0001) however not for the PNS (R2 = 0.047, p = 0.152).

Fig 2

Individual values of all eight pigs concerning optic nerve diameter, optic nerve sheath diameter and perineural space diameter for the respective ICP levels.

Left row illustrates measurements from investigator 1. Right row measurements from investigator 2.

ANOVA for repeated measurements showed a significant effect of ICP level on OND (p = 0.031) but neither a significant effect of investigator (p = 0.063), nor an interaction between the two (p = 0.089).

The median optic nerve area increased from 10.2 mm2 at baseline to 36.3 mm2 at ICP level of 60mmHg. The median optic nerve sheath area increased from 60.8 mm2 at baseline to 120.8 mm2 at ICP level of 60mmHg. The calculated perineural space area increased from 50.6 mm2 at baseline to 84.5 mm2 at ICP level of 60mmHg.

Discussion

This porcine study confirms that ultrasound measurements of the ONS diameter increase and correlate with increasing ICP. This is in line with results published in humans and pigs [2, 3, 9].

When analyzing the two different components of the ONS diameter–the optic nerve and the perineural space diameter–ONS diameter expansion can exclusively be attributed to an increase in ON diameter while the perineural space diameter shows no significant expansion. The finding that optic nerve diameter increased to a larger extent as the CSF filled perineural space diameter is unexpected. The following factors could have attributed to this finding: Capillaries and venules within the ON may have become congested. Of note, pigs have twice as many capillaries within ON compared to humans [17]. While mean arterial pressure significantly increased with increasing ICP, CPP remained unchanged up to ICP values of 50 mmHg, suggesting that cerebral perfusion probably did not increase with increasing ICP. In contrast, there is evidence that cerebral perfusion is limited by cerebral venous outflow when CPP is below 60–70 mmHg [18]. In our experiment, median CPP values were between 59 and 65 mmHg with ICP values between 20 and 50 mmHg. It is therefore conceivable that venous congestion contributed to the increase in ON diameter. Further, increasing ICP may amplify the effect of venostasis on optic nerve swelling. Diminished cerebral venous return can also be partially explained by the fact that head and the upper part of the bodies were not elevated in our pig model as opposed to standard clinical practice with head elevation in patients experiencing elevated intracranial pressure.

So far most published literature consider CSF shifts as a change detectable by ultrasound and therefore the focus is on optic nerve sheath diameter without measuring optic nerve diameter[3, 19].

However optic nerve diameter measurements have been assessed and validated. A high intra- and interobserver reliability was documented for OND measurements in healthy subjects[20]. In patients with multiple sclerosis OND can depict optic nerve atrophy[21]. Two studies conducted in patients with idiopathic intracranial hypertension found no significant difference in optic nerve diameter between control groups and patients with elevated intracranial pressure[22, 23]. However intracranial pressure levels in these studies were mildly to maximum moderately increased (ICP levels between 18 and 25 mmHg). Given the much smaller diameter of optic nerve compared with optic nerve sheath, differences might have been too small to be detected. We acknowledge, that in our study the increases in ON diameter became statistically significant only at ICP values of ≥40 mmHg. The study from Topcuoglo M et al measured different diameters within the orbit in brain death and non brain death patients[1]. Optic nerve diameter was significantly larger (p < 0.001) in brain death patients compared to non brain death patients. This finding might support our finding that also non cerebrospinal fluid changes attributed to elevated intracranial pressure can be detected by measuring optic nerve diameter using transbulbar ultrasound.

Since all measurements were obtained within minutes after the establishment of a new ICP level, the adaptive processes in cerebrospinal fluid and blood circulation may not have occurred yet. This might be particularly true for the cerebrospinal fluid circulation with its slow and steady production [24]. Despite stable perineural space diameter the area of the perineural space still increases due to the increase of inner and outer circumference of the perineural space.

Our study and therefore our findings have clear limitations. First it is an animal study with inherent problems in transmissibility to humans. The differences in optic nerve vascularization might have significantly influenced our findings. Our setup investigated only short term changes, and there is an ongoing debate about what ICP model is the most accurate one. Many different models with different advantages and disadvantaged have been proposed such as infusion of artificial CSF through a cannula placed in the subdural/subarachnoid space [25, 26], insertion and inflation of a balloon catheter into the ventricle system [26] or in the subdural space [27, 28], infusion of saline through an intra-parenchymal catheter [28, 29] and infusion of Hartmann solution in the lumbar subarachnoid space [29, 30] or reduction of cerebral venous return [9, 30]. Although we confirmed the linear increase of ONS diameter with ICP, our method was not able to indicate threshold values for increases in ICP below 40 mmHg, which limits the usefulness of the method in clinical practice.

One strength of our study is the fact that we were well able to control and obtain stable ICP levels and that we performed measurements over a very wide ICP range including very high ICP levels. Further, the investigators were blinded to the respective ICP levels when analyzing the ultrasound loops. The reasonably small coefficients of variation for inter-investigators indicate that the method used is accurate [7, 31].

Our findings should be confirmed in humans with increased ICP.

Conclusion

In a porcine ICP model, the diameter of the optic nerve correlates linearly with ICP and can be reliably measured using transbulbar ultrasonography. The increase of the ONS diameter can be mainly attributed to an increase of the ON. In contrast, the diameter of the perineural space does not correlate with ICP.

(XLSX)

Click here for additional data file.

6 Sep 2019

PONE-D-19-21626

Correlation of optic nerve and optic nerve sheath diameter with intracranial pressure in pigs

PLOS ONE

Dear Dr. Bloch,

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.

We would appreciate receiving your revised manuscript by Oct 21 2019 11:59PM. When you are 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.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

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). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Nimesh Patel

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

1. To comply with PLOS ONE submissions requirements, please provide methods of sacrifice in the Methods section of your manuscript.

Additional Editor Comments (if provided):

The work presented is important for understanding intracranial pressure. However, as pointed out by both expert reviewers, there is need to accurately describe the methodology used. Please address this major concern, along with other comments the reviewers have made.

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

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. 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: Yes

Reviewer #2: Partly

**********

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

Reviewer #1: Yes

Reviewer #2: I Don't Know

**********

3. 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: Yes

Reviewer #2: No

**********

4. 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

Reviewer #2: Yes

**********

5. 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: Dear Authors,

I enjoyed reviewing your interesting and novel work. The finding that the optic nerve diameter changes with changes in ICP is intriguing. I would like the following issues addressed that will help make this manuscript more compelling.

1. Given the known technical challenges with visualizing optic nerve anatomy in humans, it is necessary for including a figure showing the actual ultrasound images of the relevant anatomy, not just a diagram. This will give the reader the important information regarding the technical aspects of the ultrasonic technique.

2. Throughout the manuscript, please include 95% CIs whenever the p-values are reported, this will give one a better idea of the precision of the results.

3. In the introduction, pg 3, line 65. "capillaries may become congested" I am not sure I understand why the capillaries would be selectively congested, and not the venules, please clarify, and correct if indicated

4. Page 5, line 102, "left decubitus position" Do you mean, "left lateral decubitus position"?

5. Please include a picture of the experimental setup if possible for illustration

6. What methods were employed to ensure the ICP readings were accurate? Was there any calibration performed. Were the ICP waveforms evaluated for responsiveness to physiological changes in pulse and respiration to determine the accuracy and patency of the transduction system?

7. Page 10 , line 1, "perineural space" I believe this is an error and should be "optic nerve sheath" please check.

8. The discussion is inadequate with regards to the measurement of optic nerve diameter. Please elaborate on why this structure has not been measured before, or whether it has in humans and or animals in relationship to the ICP, or even as an anatomical measurement with ultrasound. Consider incorporating the article, "Chen, Q et al High-resolution transbulbar ultrasonography helping differentiate intracranial hypertension in bilateral optic disc oedema patients. Acta Ophthalmologica. 2017

9. page 9, "Further, elevated ICP levels cause additional vasogenic edema..." I do not understand the physiological mechanism referred to here. Vasogenic edema does not accompany all ICP elevation, especially if the ICP elevation does not disrupt the blood brain barrier, consider omitted this statement or clarifying it.

10.

Please correct multiple typographical errors for example...

pg 4, line 83, "intracompartimental"

Pg 9, line 8, "extend" should be "extent"

pg 9, "liquor" is usually referred to cerebrospinal fluid, please correct

Reviewer #2: Thanks for the opportunity to review this article. Since the entire work is relying on ultrasound image-based measurement values, it is imperative that representative images are shown, along with the measurement points. This is even more important since 40 kg porcine optic nerve at baseline should be smaller (human ON rarely exceeds 3 mm). The diagram (Figure 1) is clearly insufficient.

Porcine optic nerve imaging is very difficult due to a number of impediments, including positional instability of the eyes, near-impossibility to bypass the large lens, limited probe positioning options, and others. These difficulties, combined with unexpected results, make it even more important for the reader to see the actual images of the measured structures with the measuring technique.

**********

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: Yes: Eric Bershad

Reviewer #2: 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 to be viewed.]

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 us at figures@plos.org. Please note that Supporting Information files do not need this step.

23 Oct 2019

Editor comments

To comply with PLOS ONE submissions requirements, please provide methods of sac-rifice in the Methods section of your manuscript.

Response

In order to comply with PLOS ONE submissions requirements we have added details concerning sacrifice of the pigs in the methods section. The following sentence was added. “… At the end of the experiment a bolus of 40 mmol potassium chloride was given to sacrifice the animal.”

Reviewer #1:

Dear Authors,

I enjoyed reviewing your interesting and novel work. The finding that the optic nerve diameter changes with changes in ICP is intriguing. I would like the following issues addressed that will help make this manuscript more compelling.

Reviewer comment

1. Given the known technical challenges with visualizing optic nerve anatomy in hu-mans, it is necessary for including a figure showing the actual ultrasound images of the relevant anatomy, not just a diagram. This will give the reader the important infor-mation regarding the technical aspects of the ultrasonic technique.

Response

We have included the following figure showing two actual ultrasound images with the relevant anatomy marked with arrows and stars. One image was recorded at baseline ICP (9 mmHg) and the second one at an ICP of 40mmHg. We have therefore excluded the previous schematic figure 1. This will hopefully improve the illustration of the tech-nical aspects.

Fig 1: The upper transbulbar ultrasound image was recorded at baseline ICP – the lower image was recorded at ICP of 40mmHg. All measurements were obtained 3 mm behind the papilla as indicated by the two stars connected with the dotted line. Optic nerve diameter increased from 0.36 cm to 0.48 cm as illustrated by the black arrow. Op-tic nerve sheath diameter increased from 0.79 cm to 0.86 cm as illustrated by the white arrow.

Reviewer comment

2. Throughout the manuscript, please include 95% CIs whenever the p-values are re-ported, this will give one a better idea of the precision of the results.

Response

We have followed your valid input and we have added the respective 95% confidence intervals when p- values were reported.

Reviewer comment

3. In the introduction, pg 3, line 65. "capillaries may become congested" I am not sure I understand why the capillaries would be selectively congested, and not the venules, please clarify, and correct if indicated

Response

Thanks to your input we have corrected the obviously physiological inprecise state-ment “capillaries may become congested” to “capillaries and venules within ON may become congested…”

Reviewer comment

4. Page 5, line 102, "left decubitus position" Do you mean, "left lateral decubitus posi-tion"?

Reponse

We have revised the term “left decubitus position” to “left lateral decubitus position”.

Reviewer comment

5. Please include a picture of the experimental setup if possible for illustration

Response

We did not take any picture during the trial so we cannot accommodate this demand.

Reviewer comment

6. What methods were employed to ensure the ICP readings were accurate? Was there any calibration performed. Were the ICP waveforms evaluated for responsiveness to physiological changes in pulse and respiration to determine the accuracy and patency of the transduction system?

Response

For ICP-monitoring a bolt-kit silver-bearing EVD catheter system (Silverline-Bolt-Kit-catheter; Spiegelberg GmbH & Co. KG; Hamburg, Germany) was implanted. This cathe-ter type is equipped with an intraparenchymal/intraventricular ICP measurement sys-tem via an air-pouch mounted close to the tip of the drainage catheter. The air-pouch is connected via a separate lumen to a standalone ICP monitor. This monitor itself is connected to the multi-modular monitor (S/5 Critical Care Monitor®; Datex-Ohmeda, GE Healthcare, Helsinki, Finland) where the ICP waveform is displayed. The Spiegel-berg ICP measurement system has an automatic calibration algorhythm and a ne-glectable drift over time. In addition, conventional ICP measurement is also possible via the drainage catheter. After implantation of the EVD catheter, the ICP measured with the Spiegelberg monitor was compared with the conventional ICP measurement via the CSF coupled pressure transducer to check for accuracy. Furthermore, the ICP waveform was evaluated for responsiveness to manoevers reducing the venous back flow. For the further experimental procedures, the Spiegelberg ICP monitoring system was used.

Reviewer comment

7. Page 10 , line 1, "perineural space" I believe this is an error and should be "optic nerve sheath" please check.

Response

Given that the effective area (see calculations below) of the perineural space increas-es, the statement is correct. However in order to clarify and specify this we have changed the sentence from “Despite stable perineural space diameter the area of the perineural space still increases due to the overall increase of the combined diameters of the ON and perineural space” to “Despite stable perineural space diameter the area of the perineural space still increases due to the increase of inner and outer circumfer-ence of the perineural space” .

Calculations:

Calculating the areas of the components ON and ONS from baseline to values at 60 mmHg using the formula area = π / diameter 2 results in the following values:

Area ON (optic nerve) at baseline = 10.2 mm2

Area ON (optic nerve) at ICP 60 mmHg = 36.3 mm2

Area ONS (optic nerve sheath) at baseline = 60.8 mm2

Area ONS (optic nerve sheath) at 60 mmHg = 120.8 mm2

The area of the perineural space can be calculated by subtracting the optic nerve area from the optic nerve sheath area. This results in the following values:

Area PNS (perineural space) at baseline = 50.6 mm2

Area PNS (perineural space) at 60 mmHg = 84.5 mm2

We have further tried to emphasize this finding by integrating the calculation and the results of the respective changes of the different areas into the manuscript.

Reviewer comment

8. The discussion is inadequate with regards to the measurement of optic nerve diame-ter. Please elaborate on why this structure has not been measured before, or whether it has in humans and or animals in relationship to the ICP, or even as an anatomical measurement with ultrasound. Consider incorporating the article, "Chen, Q et al High-resolution transbulbar ultrasonography helping differentiate intracranial hypertension in bilateral optic disc oedema patients. Acta Ophthalmologica. 2017

Response

Thank you for providing this interesting reference article. It is in fact difficult to find op-tic nerve diameter measurements in articles concerning transbulbar sonography. It is somewhat hard to elaborate the reasons for that. The most obvious explanation might be that no difference in optic nerve diameter was found which was also the case in the paper from “Chen et al”. Most studies on transbulbar sonography were conducted in patients with mild to maximally moderately increased ICP levels. Given the much smaller diameter of the optic nerve compared with the optic nerve sheath differences might be missed in these ICP ranges. In the present study, the increase in ON was significant only with ICP levels ≥40 mmHg Further, most authors only considered the cerebrospinal fluid shifts as a change detectable by ultrasound and they therefore fo-cused on optic nerve sheath diameter changes only.

The article from “Topcuoglo M et al, Transorbital Ultrasonographic Measurements of Optic Nerve Sheath Diameter in Brain Death. Journal Neuroimaging. 20151” measured optic nerve diameter and optic nerve sheath diameter in brain-death and non brain-death patients. Optic nerve diameter was significantly larger (p < 0.001) in brain-death patients compared to non brain-death patients. This finding might support our finding that also non cerebrospinal fluid changes attributed to elevated intracranial pressure can be detected by transbulbar ultrasound by measuring optic nerve diameter.

We have adjusted the discussion part of the manuscript. The abstract related to your comment reads as follows:

So far most published literature consider CSF shifts as a change detectable by ultra-sound and therefore the focus is on optic nerve sheath diameter without measuring optic nerve diameter2,3.

However optic nerve diameter measurements have been assessed and validated. A high intra- and interobserver reliability was documented for OND measurements in healthy subjects4. In patients with multiple sclerosis OND can depict optic nerve atro-phy5. Two studies conducted in patients with idiopathic intracranial hypertension found no significant difference in optic nerve diameter between control groups and patients with elevated intracranial pressure6,7. However intracranial pressure levels in these studies were mildly to maximum moderately increased (ICP levels between 18 and 25 mmHg). Given the much smaller diameter of optic nerve compared with optic nerve sheath, differences might have been too small to be detected. We acknowledge, that in our study the increases in ON diameter became statistically significant only at ICP values of ≥40 mmHg. The study from Topcuoglo M et al measured different diame-ters within the orbit in brain death and non brain death patients1. Optic nerve diameter was significantly larger (p < 0.001) in brain death patients compared to non brain death patients. This finding might support our finding that also non cerebrospinal fluid changes attributed to elevated intracranial pressure can be detected by measuring op-tic nerve diameter using transbulbar ultrasound.

Reviewer comment

9. page 9, "Further, elevated ICP levels cause additional vasogenic edema..." I do not understand the physiological mechanism referred to here. Vasogenic edema does not accompany all ICP elevation, especially if the ICP elevation does not disrupt the blood brain barrier, consider omitted this statement or clarifying it.

Response

We have omitted this statement due to your valid comment. Thank you.

Reviewer comment

10.

Please correct multiple typographical errors for example...

pg 4, line 83, "intracompartimental"

Pg 9, line 8, "extend" should be "extent"

pg 9, "liquor" is usually referred to cerebrospinal fluid, please correct

Response

We have thoroughly reviewed the manuscript with regards to typographical errors. We thankfully integrated your corrections.

Reviewer #2:

Reviewer comment

Reviewer #2: Thanks for the opportunity to review this article. Since the entire work is relying on ultrasound image-based measurement values, it is imperative that repre-sentative images are shown, along with the measurement points. This is even more important since 40 kg porcine optic nerve at baseline should be smaller (human ON rarely exceeds 3 mm). The diagram (Figure 1) is clearly insufficient.

Porcine optic nerve imaging is very difficult due to a number of impediments, including positional instability of the eyes, near-impossibility to bypass the large lens, limited probe positioning options, and others. These difficulties, combined with unexpected results, make it even more important for the reader to see the actual images of the measured structures with the measuring technique.

Response

Thank you for your valid comment.

We have included the following figure showing two actual ultrasound images with the relevant anatomy marked with arrows and stars. One image was recorded at baseline ICP (9 mmHg) and the second one at an ICP of 40mmHg. We have therefore excluded the previous schematic figure 1.

As pointed out in the discussion our findings and our setup have clear limitations in-cluding inherent problems in transmissibility to humans. The differences in optic nerve vascularization might have significantly influenced our findings. However our setup with stable left lateral decubitus position and deep sedation of the animals might have facilitated the difficulties that you had pointed out. Hopefully the current figure will help the reader to better understand and to be able to relate to the described method.

Fig 1: The upper transbulbar ultrasound image was recorded at baseline ICP – the lower image was recorded at ICP of 40mmHg. All measurements were obtained 3 mm behind the papilla as indicated by the two stars connected with the dotted line. Optic nerve diameter increased from 0.36 cm to 0.48 cm as illustrated by the black arrow. Op-tic nerve sheath diameter increased from 0.79 cm to 0.86 cm as illustrated by the white arrow.

References

1. Topcuoglu MA, Arsava EM, Bas DF, Kozak HH. Transorbital Ultrasonographic Measurement of Optic Nerve Sheath Diameter in Brain Death. Journal of neuroimaging : official journal of the American Society of Neuroimaging. 2015;25(6):906-909.

2. Dubourg J, Javouhey E, Geeraerts T, Messerer M, Kassai B. Ultrasonography of optic nerve sheath diameter for detection of raised intracranial pressure: a systematic review and meta-analysis. Intensive care medicine. 2011;37(7):1059-1068.

3. Fernando SM, Tran A, Cheng W, et al. Diagnosis of elevated intracranial pressure in critically ill adults: systematic review and meta-analysis. Bmj. 2019;366:l4225.

4. Lochner P, Coppo L, Cantello R, et al. Intra- and interobserver reliability of transorbital sonographic assessment of the optic nerve sheath diameter and optic nerve diameter in healthy adults. Journal of ultrasound. 2016;19(1):41-45.

5. Candeliere Merlicco A, Gabaldon Torres L, Villaverde Gonzalez R, Fernandez Romero I, Aparicio Castro E, Lastres Arias MC. Transorbital ultrasonography for measuring optic nerve atrophy in multiple sclerosis. Acta neurologica Scandinavica. 2018;138(5):388-393.

6. Chen Q, Chen W, Wang M, et al. High-resolution transbulbar ultrasonography helping differentiate intracranial hypertension in bilateral optic disc oedema patients. Acta ophthalmologica. 2017;95(6):e481-e485.

7. Jeub M, Schlapakow E, Ratz M, et al. Sonographic assessment of the optic nerve and the central retinal artery in idiopathic intracranial hypertension. Journal of clinical neuroscience : official journal of the Neurosurgical Society of Australasia. 2019.

11 Nov 2019

PONE-D-19-21626R1

Correlation of optic nerve and optic nerve sheath diameter with intracranial pressure in pigs

PLOS ONE

Dear Dr. Bloch,

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.

We would appreciate receiving your revised manuscript by Dec 26 2019 11:59PM. When you are 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.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

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). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Nimesh Patel

Academic Editor

PLOS ONE

Additional Editor Comments (if provided):

Thank you for your revisions. Please address carefully the requested change by the second reviewer.

[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: All comments have been addressed

Reviewer #2: (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: Yes

Reviewer #2: Partly

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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: Yes

Reviewer #2: No

**********

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: Yes

Reviewer #2: Yes

**********

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: (No Response)

Reviewer #2: Thanks for replacing a diagram with actual ultrasound images. However, these provided images are not much superior compared to the diagram. While a slight variation of the imaging plane is acceptable, I find it disappointing that only fragments of the images are shown. The probe that you are using is excellent for this purpose, and could probably allow bypassing the lens; Judging by the similarity and quality of the patterns in the two images, it seems like the lens was indeed bypassed (or at least, transected with a similar aberration). But the reader should not have to guess (or remain oblivious to the fact that the lens may introduce major distortions and affect the measurements of the posterior structures). The reader does not learn from these images, nor do these images serve to assure the reader of proper technique hence accurate results. I would strongly recommend providing full images with a depth scale, otherwise my concerns are not alleviated.

**********

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: Yes: Eric Bershad

Reviewer #2: 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 to be viewed.]

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 us at figures@plos.org. Please note that Supporting Information files do not need this step.

25 Dec 2019

Editor comments

Thank you for your revisions. Please address carefully the requested change by the second reviewer.

Response

Thank you for the possibility to address this important issue recognised by the second reviewer. The figure 1 has been replaced by a new figure 1.

Reviewer #2:

Thanks for replacing a diagram with actual ultrasound images. However, these provid-ed images are not much superior compared to the diagram. While a slight variation of the imaging plane is acceptable, I find it disappointing that only fragments of the imag-es are shown. The probe that you are using is excellent for this purpose, and could probably allow bypassing the lens; Judging by the similarity and quality of the patterns in the two images, it seems like the lens was indeed bypassed (or at least, transected with a similar aberration). But the reader should not have to guess (or remain oblivious to the fact that the lens may introduce major distortions and affect the measurements of the posterior structures). The reader does not learn from these images, nor do these images serve to assure the reader of proper technique hence accurate results. I would strongly recommend providing full images with a depth scale, otherwise my concerns are not alleviated.

Response

Thank you for this very valid comment. We have followed your recommendation and we have provided the full images with a depth scale. As you can see both images rep-resent images made from high definition zooms with a zoom box size of 2 cm – there-fore the depth scale does not start at 0.

In both ultrasound images the lenses were indeed bypassed and therefore no major distorsions were caused by the lens nor were the measurements affected.

We have further supplemented the legend of the figure as follows:

The upper transbulbar ultrasound image was recorded at baseline ICP – the lower im-age was recorded at ICP of 40mmHg. All measurements were obtained 3 mm behind the papilla as indicated by the two stars connected with the dotted line. Optic nerve di-ameter increased from 0.36 cm to 0.48 cm as illustrated by the black arrow. Optic nerve sheath diameter increased from 0.79 cm to 0.86 cm as illustrated by the white arrow.

In both ultrasound images the lens was bypassed in order to obtain the most accurate display of the posterior structures of the eye. Further both images were made by using a high definition zoom box of 2 cm size.

13 Jan 2020

Correlation of optic nerve and optic nerve sheath diameter with intracranial pressure in pigs

PONE-D-19-21626R2

Dear Dr. Bloch,

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

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. 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 enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and 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.

With kind regards,

Nimesh Patel

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Thank you for addressing all concerns of the reviewers.

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: All comments have been addressed

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 #1: Yes

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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: Yes

Reviewer #2: Yes

**********

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: Yes

Reviewer #2: Yes

**********

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: (No Response)

Reviewer #2: Thanks for clarifications and a detailed response to reviewer concerns. Congratulations with successful completion of a technically challenging experiment. Best wishes to the team.

**********

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: Yes: Eric Bershad

Reviewer #2: No

21 Jan 2020

PONE-D-19-21626R2

Correlation of optic nerve and optic nerve sheath diameter with intracranial pressure in pigs

Dear Dr. Bloch:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. 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.

For any other questions or concerns, please email plosone@plos.org.

Thank you for submitting your work to PLOS ONE.

With kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Nimesh Patel

Academic Editor

PLOS ONE