Confocal and Electron Microscopic Structure of the Cornea from Three Species of Penguin

Round 1

Reviewer 1 Report

This work reports the study of corneas from three species of penguin using confocal and electron microscopy.

Please give more details in the preparation for the confocal study of the corneas. For example, refrigeration temperature (please specify the temperature, like 4 degree), and state how the sample was thawed if any freezing was done (if there was any). Please also state the medium in which the sample was kept. Moreover, please describe how to achieve focusing of image since a working distance of 0.0 mm, relative to the applanating cap, was used. This discussion is necessary because compression artifacts could be easily produced which could affect the density of cells.   

Please justify the reason for the different number of penguins used for the different species in this study. Additionally, please provide the reason for no fresh sample investigated in Gentoo penguins. Similarly, explain the reason for the different treatment employed for the various samples.

According to the author, “fresh” sample defines as “the ocular examination was completed within 6 hours of death, without being stored in any medium” (line 99-100). Nevertheless, please specify the refractive index matching medium in which the sample placed during the confocal microscopy imaging, since water immersion lens was used.

Author Response

Thank you for your review. We have incorporated all of your suggestions in our revised manuscript, as below:

Comment 1:

Please give more details in the preparation for the confocal study of the corneas. For example, refrigeration temperature (please specify the temperature, like 4 degree), and state how the sample was thawed if any freezing was done (if there was any). Please also state the medium in which the sample was kept. Moreover, please describe how to achieve focusing of image since a working distance of 0.0 mm, relative to the applanating cap, was used. This discussion is necessary because compression artifacts could be easily produced which could affect the density of cells.  

We have discussed this in much greater detail, changing the relevant paragraph in the methods to the below. We also added the temperature to the table

All eyes were enucleated following euthanasia. Some were transported directly to the laboratory in a plastic container filled only with air (‘Fresh’ in Table 1). Where we anticipated a longer period of time between enucleation and examination, the enucleated eyes were stored either in normal saline, Optisol culture medium or air (i.e. without a storage medium) and refrigerated at 4˚C. Confocal microscopy was performed on the cornea using the Heidelberg Retina Tomograph III (HRTIII) Rostock Corneal Module (RCM) (Heidelberg Engineering GmBH, Germany). The HRTIII is a laser scanning in vivo confocal microscope (IVCM), utilising a coherent Helium Neon diode laser (670 nm). The HRTIII also has a coupled CCD camera lateral to the IVCM, allowing the operator to monitor the position of the HRTIII relative to the cornea. With the 60x objective water immersion lens with a numerical aperture of 0.9 (Olympus, Tokyo, Japan) the IVCM images that are produced are 400 x 400 μm in dimension with a digital image size of 384 x 384 pixels (1.04 μm/pixel), a lateral resolution of 2 μm and optical section thickness of 4 μm. The applanating cap of the HRTIII must applanate (be in contact with) the cornea in order to image the corneal microstructure. Once applanation is achieved, the depth at which the optical section is produced is altered by adjusting the focusing head of the IVCM.  As the device is an IVCM, the eye and lens are not immersed in water, instead, Viscotears (Carbomer 980, 0.2%, Novartis, Australia) was applied to the eye and used as a coupling agent between the applanating lens cap and the cornea. Eyes were held in place by hand which in turn were resting on the chin rest of the device to maintain a stable position during image capture The full thickness of the central cornea was scanned using the device’s “section mode”. The section mode enables instantaneous imaging of a single area of the cornea at a desired depth. Images were captured continuously to ensure at least three compression free images of each layer were obtained. All images were reviewed and analysed by an experienced examiner (AG) and only compression free images were utilised. Measurements were performed using the proprietary RCM software and cells were counted only if they were readily identifiable rather than a smudge, the implication being that they are in the same plane. Because of variable cellular density (Fig. 1B), three different images were counted at each layer of each cornea that was examined, the layers being superficial epithelium, basal epithelium, anterior, mid- and posterior stroma as well as the endothelium.

Comment 2.

Please justify the reason for the different number of penguins used for the different species in this study. Additionally, please provide the reason for no fresh sample investigated in Gentoo penguins. Similarly, explain the reason for the different treatment employed for the various samples.

We have added the following sentence:

The sample size was the result of the number of penguins being euthanized over the study period of three years; little penguins found dead in the wild were not sought because of potential post-mortem changes.

Comment 3.

According to the author, “fresh” sample defines as “the ocular examination was completed within 6 hours of death, without being stored in any medium” (line 99-100). Nevertheless, please specify the refractive index matching medium in which the sample placed during the confocal microscopy imaging, since water immersion lens was used.

We have done this in the same paragraph as we addressed comment 1.

Reviewer 2 Report

The authors presented a interesting study on the confocal and electron microscopic structure of the cornea from three species of penguin. The manuscript is with merit but the following comments should be addressed:

-       Introduction

o   What do the authors mean by “more powerful” corneas? Are they referring to the shape? Please provide details

o   The last paragraph of the introduction should be moved to the discussion section. The last paragraph of the introduction should be focused on the aim of the study, not on the results/discussion of the study itself

 

-       Methods

o   In the methods the authors should provide some justification/power statement of the sample size of the study

Author Response

Thank you for your review. We have incorporated all of your suggestions in our revised manuscript, as below:

Comment 1.

What do the authors mean by “more powerful” corneas? Are they referring to the shape? Please provide details

Yes this was sloppy; we were referring to shape and of course dioptric power is not merely just the result of shape. Therefore we have changed this to:

However, the shape of the penguin cornea does vary, with the smaller penguins having smaller, more steeply curved corneas [3].

Comment 2.

The last paragraph of the introduction should be moved to the discussion section. The last paragraph of the introduction should be focused on the aim of the study, not on the results/discussion of the study itself

We have done this. In fact, after looking more closely we deleted almost the entire paragraph as it was almost totally repeated in the discussion section. We added only "Conversely, the limbus appeared very similar to that of humans." to the discussion.

Comment 3.

In the methods the authors should provide some justification/power statement of the sample size of the study

We have added:

The sample size was the result of the number of penguins being euthanized over the study period of three years; little penguins found dead in the wild were not sought because of potential post-mortem changes.

Reviewer 3 Report

The paper entitled “Confocal and electron microscopic structure of the cornea from three species of penguin” is a study based on corneal confocal microscopy performed on different species of penguins. The study plan is quite interesting and innovative. The data is based on histological specimens and performed in a prospective manner. The data collected surely adds to the literature in this field, which is lacking. The results regarding corneal characteristics and histology can provide information about adaptation and evolutionary processes in penguins.

The study has been correctly planned. It is well-written and of clinical interest. The study provides objective results, which add to the current literature in this field. Figures are pertinent, and descriptive and assist in describing the results.

Author Response

Thank you for review. We have made some changes as suggested by reviewers 1 and 2.