Author response:
The following is the authors’ response to the previous reviews
Reviewer #1 (Public review):
Summary:
The authors aimed to investigate the interaction between tissue-resident immune cells (microglia) and circulating systemic neutrophils in response to acute, focal retinal injury. They induced retinal lesions using 488 nm light to ablate photoreceptor (PR) outer segments, then utilized various imaging techniques (AOSLO, SLO, and OCT) to study the dynamics of fluorescent microglia and neutrophils in mice over time. Their findings revealed that while microglia showed a dynamic response and migrated to the injury site within a day, neutrophils were not recruited to the area despite being nearby. Post-mortem confocal microscopy confirmed these in vivo results. The study concluded that microglial activation does not recruit neutrophils in response to acute, focal photoreceptor loss, a scenario common in many retinal diseases.
Strengths:
The primary strength of this manuscript lies in the techniques employed.
In this study, the authors utilized advanced Adaptive Optics Scanning Laser Ophthalmoscopy (AOSLO) to document immune cell interactions in the retina accurately. AOSLO's micron-level resolution and enhanced contrast, achieved through near-infrared (NIR) light and phase-contrast techniques, allowed visualization of individual immune cells without extrinsic dyes. This method combined confocal reflectance, phase-contrast, and fluorescence modalities to reveal various cell types simultaneously. Confocal AOSLO tracked cellular changes with less than 6 μm axial resolution, while phase-contrast AOSLO provided detailed views of vascular walls, blood cells, and immune cells. Fluorescence imaging enabled the study of labeled cells and dyes throughout the retina. These techniques, integrated with conventional histology and Optical Coherence Tomography (OCT), offered a comprehensive platform to visualize immune cell dynamics during retinal inflammation and injury.
Thank you!
Weaknesses:
One significant weakness of the manuscript is the use of Cx3cr1GFP mice to specifically track GFP-expressing microglia. While this model is valuable for identifying resident phagocytic cells when the blood-retinal barrier (BRB) is intact, it is important to note that recruited macrophages also express the same marker following BRB breakdown. This overlap complicates the interpretation of results and makes it difficult to distinguish between the contributions of microglia and infiltrating macrophages, a point that is not addressed in the manuscript.
We agree that greater emphasis is required that CX3CR1 mice exhibit fluorescence in not only microglia, but also other cells of macrophage origin including monocytes, perivascular macrophages and some hyalocytes.
Through the advantages of in vivo AOSLO, however, we are able to establish that CX3CR1 cells are present within the tissue before the laser lesion is placed. This suggests they are tissue resident. We agree that it is possible that at later time points (days-weeks), systemic macrophages and/or monocytes may participate. Lack of rolling/crawling cells suggest they are not systemic. We elaborate on this point in a new section in the discussion:
P29 L534-541:
“CX3CR1-GFP mice exhibit fluorescence not only in microglia
We recognize that the CX3CR1-GFP model can also label systemic cells such as monocytes/macrophages77. While it is possible these cells could infiltrate the retina in response to the lesion, we find it unlikely since there was no indication of the leukocyte extravasation cascade (rolling/crawling/stalled cells) within the nearest retinal vasculature. In addition to microglia, retinal perivascular macrophages and hyalocytes also exhibit GFP fluorescence and thus that these cells may also contribute toward damage resolution.”
Another major concern is the time point chosen for analyzing the neutrophil response. The authors assess neutrophil activity 24 hours after injury, which may be too late to capture the initial inflammatory response. This delayed assessment could overlook crucial early dynamics that occur shortly after injury, potentially impacting the overall findings and conclusions of the study.
The power of in vivo imaging makes these early assessments possible. Therefore, we have taken the reviewers concern and conducted an additional experiment which examines whether neutrophils are seen in the window of time between lesion and 24hrs. In a newly examined mouse, we find that within 3.5 hours post-lesion, neutrophils do not extravasate adjacent to the lesion site (see new “figure 8 – figure supplement 1”).
Also see accompanying video (new “figure 8 – video 3”) for an example of nearby neutrophils flowing through OPL capillaries just microns away from the lesion site. Neutrophils are clearly contained within the vasculature and exhibit dynamics consistent with healthy retinal tissue. While it remains possible that the lesion may increase leukocyte stalling within the nearest capillaries, we are unable to confirm or deny this with a single experiment. We now submit this evidence as a new supplementary figure following the reviewer’s suggestion.
Reviewer #2 (Public review):
Summary:
This study uses in vivo multimodal high-resolution imaging to track how microglia and neutrophils respond to light-induced retinal injury from soon after injury to 2 months post-injury. The in vivo imaging finding was subsequently verified by an ex vivo study. The results suggest that despite the highly active microglia at the injury site, neutrophils were not recruited in response to acute light-induced retinal injury.
Strengths:
An extremely thorough examination of the cellular-level immune activity at the injury site. In vivo imaging observations being verified using ex vivo techniques is a strong plus.
We appreciate this recognition and hope that the reviewer considers the weaknesses below in the context of the papers identified strengths.
Weaknesses:
This paper is extremely long, and in the perspective of this reviewer, needs to be better organized.
We agree and have taken the following steps to address this:
(1) Paper has been shortened overall by 8%
(2) We reorganized the following sections:
a. Introduction: shortened
b. Methods: merged section “Ex vivo confocal image processing” with “Ex vivo confocal imaging”.
c. Results: most sections shortened, others simplified for concision
d. Discussion: most sections shortened, removed “Microglial/neutrophil discrimination using label-free phase contrast”
e. Figure references reorganized in order of their appearance.
Study weakness: though the finding prompts more questions and future studies, the findings discussed in this paper are potentially important for us to understand how the immune cells respond differently to different severity levels of injury.
On the heels of this burgeoning technology, we consider this report among the first studies of its kind. We are hopeful that it forms the foundation of many further investigations to come. We expect a rich parameter space to be explored with future studies including investigation of other time points, other injuries of varying degree and other immune cell populations (along with their interactions with each other). Each has the potential to reveal the complexities of the ocular immune system in action.
Reviewer #3 (Public review):
Summary:
This work investigated the immune response in the murine retina after focal laser lesions. These lesions are made with close to 2 orders of magnitude lower laser power than the more prevalent choroidal neovascularization model of laser ablation. Histology and OCT together show that the laser insult is localized to the photoreceptors and spares the inner retina, the vasculature, and the pigment epithelium. As early as 1-day after injury, a loss of cell bodies in the outer nuclear layer is observed. This is accompanied by strong microglial proliferation at the site of injury in the outer retina where microglia do not typically reside. The injury did not seem to result in the extravasation of neutrophils from the capillary network constituting one of the main findings of the paper. The demonstrated paradigm of studying the immune response and potentially retinal remodeling in the future in vivo is valuable and would appeal to a broad audience in visual neuroscience. However, there are some issues with the conclusions drawn from the data and analysis that can be addressed to further bolster the manuscript.
Strengths:
Adaptive optics imaging of the murine retina is cutting edge and enables non-destructive visualization of fluorescently labeled cells in the milieu of retinal injury. As may be obvious, this in vivo approach is beneficial for studying fast and dynamic immune processes on a local time scale - minutes and hours, and also for the longer days-to-months follow-up of retinal remodeling as demonstrated in the article. In certain cases, the in vivo findings are corroborated with histology.
Thank you!
The analysis is sound and accompanied by stunning video and static imagery. A few different sets of mouse models are used, (a) two different mouse lines, each with a fluorescent tag for neutrophils and microglia, (b) two different models of inflammation - endotoxin-induced uveitis (EAU) and laser ablation are used to study differences in the immune interaction.
Thank you!
One of the major advances in this article is the development of the laser ablation model for 'mild' retinal damage as an alternative to the more severe neovascularization models. While not directly shown in the article, this model would potentially allow for controlling the size, depth, and severity of the laser injury opening interesting avenues for future study.
We agree that there is an established community that is invested in developing titrated dosimetry for light damage models. As the reviewer recognizes, this parameter space is exceptionally large therefore we controlled this parameter by choosing a single wavelength that is commonly used in ophthalmoscopy (488nm), fixed duration and exposure regime that created a reproducible, mild damage of photoreceptors. At this titration we created a mild lesion that spares retina above and below.
Weaknesses:
(1) It is unclear based on the current data/study to what extent the mild laser damage phenotype is generalizable to disease phenotypes. The outer nuclear cell loss of 28% and a complete recovery in 2 months would seem quite mild, thus the generalizability in terms of immune-mediated response in the face of retinal remodeling is not certain, specifically whether the key finding regarding the lack of neutrophil recruitment will be maintained with a stronger laser ablation.
It seems the concern here is whether our finding is generalizable to other damage regimes, especially more severe ones. While speculative, we would suspect that it is not generalizable across different lesions of greater severity. For example, puncturing Bruch’s membrane is an example of a more severe phenotype that is often encountered in laser damage. However, this creates a complicated model that not only induces inflammation, but also compromises BRB integrity and promotes CNV. The parameter space to be tested in the reviewer’s question is quite vast and therefore have tried to summarize the generalizability within our manuscript in
P31 L586-588 “There are limitations on how generalizable this mild damage to more severe damage or disease phenotypes, but this acute damage model can begin to provide clues about how immune cells interact in response to PR loss. In this laser lesion model, we ablate 27% of the PRs in a 50 µm region.”
(2) Mice numbers and associated statistics are insufficient to draw strong conclusions in the paper on the activity of neutrophils, some examples are below:
a) 2 catchup mice and 2 positive control EAU mice are used to draw inferences about immune-mediated activity in response to injury. If the goal was to show 'feasibility' of imaging these mouse models for the purposes of tracking specific cell type behavior, the case is sufficiently made and already published by the authors earlier. It is possible that a larger sample size would alter the conclusion.
We would like to highlight that the total number of mice studied in this report was 28 (18 in-vivo imaging, 10 ex-vivo histology, >40 lesions total). While power analysis is challenging as these are the first studies of their kind, we underscore that in vivo imaging allows those same mice to be studied multiple times longitudinally. This is not possible with traditional histology. Therefore, in vivo imaging not only reveals the temporal progression (unlike histology), but also increases the number of observations beyond a simple count of the “number of mice”.
The goal of the study was not one of feasibility. The goal was to address a specific question in ocular biology: “do resident CX3CR1 cells recruit neutrophils in early, regional retinal injury”
The low numbers that the reviewer points to, are not the primary data of the paper, rather, supportive control data. Moreover, we refocus the attention on the fact that our study is performed on 28 mice across multiple modalities and each corroborates a common finding that neutrophils do not appear to be recruited despite strong microglial response; a central finding of the paper.
b) There are only 2 examples of extravasated neutrophils in the entire article, shown in the positive control EAU model. With the rare extravasation events of these cells and their high-speed motility, the chance of observing their exit from the vasculature is likely low overall, therefore the general conclusions made about their recruitment or lack thereof are not justified by these limited examples shown.
The spirit of the challenge raised is that because nothing was seen, is not proof that nothing occurred. Said more commonly, “absence of evidence is not evidence of absence”- a quote often attributed to Carl Sagan. Yet we push back on this conjecture as we have shown, not only with cutting edge in vivo imaging, but also with ample histological controls as well as multiple transgenic animals (and corroborating IHC antibodies) that in none of these imaging modalities, at none of the time points we evaluated, did neutrophils aggregate or extravasate in response to photoreceptor ablation.
Reviewer adds: “the chance of observing their exit from the vasculature is likely low overall…”
This is the reason that we specifically chose a focal lesion model to increase any possible chance of imaging a rare event. The focal lesion provides both a time and a location for “where” to look. Small 50 micrometer lesions were sufficient to drive a strong local microglial response (figures 5,6,9). This was evidence that local inflammatory cues were present. Yet despite this activation, neutrophils were not recruited to this location. We emphasize that this is a strength of our approach over other pan-retinal damage models that may indeed miss the rare extravasation events that are geographically sparse and happen over hours.
c) In Figure 3, the 3-day time point post laser injury shows an 18% reduction in the density of ONL nuclei (p-value of 0.17 compared to baseline). In the case of neutrophils, it is noted that "Control locations (n = 2 mice, 4 z-stacks) had 15 {plus minus} 8 neutrophils per sq.mm of retina whereas lesioned locations (n = 2 mice, 4 z-stacks) had 23 {plus minus} 5 neutrophils per sq.mm of retina (Figure 10b). The difference between control and lesioned groups was not statistically significant (p = 0.19)." These data both come from histology. While the p-values - 0.17 and 0.19 - are similar, in the first case a reduction in ONL cell density is concluded while in the latter, no difference in neutrophil density is inferred in the lesioned case compared to control. Why is there a difference in the interpretation where the same statistical test and methodology are used in both cases? Besides this statistical nuance, is there an alternate possibility that there is an increased, albeit statistically insignificant, concentration of circulating neutrophils in the lesioned model? The increase is nearly 50% (15 {plus minus} 8 vs. 23 {plus minus} 5 neutrophils per sq.mm) and the reader may wonder if a larger animal number might skew the statistic towards significance.
The statistics and p-values will be dependent on the strategy of analysis performed. As described in the methods, we used a predetermined 50 micron cylinder for our counting analysis based on the average lesion size created. We used this circular window to roughly approximate the size of the common lesion size. However, recall that the damage is created in a single axis (a line projected on the retina) therefore it is possible that the analysis region is too generous to capture the exceptionally local damage.
While the reviewer is focused on the nuance of statistics, we would like to refocus the conversation on our data that shows that very few neutrophils were observed at all (105 cells from 8 locations, P value reported). But missed in the above critique is that all neutrophils were contained within capillaries (Fig 10). We found no examples of extravasated neutrophils. This is the major finding and is supported by our in vivo as well as ex vivo confirmation.
(2) The conclusions on the relative activity of neutrophils and microglia come from separate animals. The reader may wonder why simultaneous imaging of microglia and neutrophils is not shown in either the EAU mice or the fluorescently labeled catchup mice where the non-labeled cell type could possibly be imaged with phase-contrast as has been shown by the authors previously. One might suspect that the microglia dynamics are not substantially altered in these mice compared to the CX3CR1-GFP mice subjected to laser lesions, but for future applicability of this paradigm of in vivo imaging assessment of the laser damage model, including documenting the repeatability of the laser damage model and the immune cell behavior, acquiring these data in the same animals would be critical.
A double fluorescent mouse (neutrophils and microglia) is a logical next step of this research. In fact, we have now crossed these transgenic mice and are studying this double labeled mouse in a second manuscript in preparation. However, for this study, it was imperative that the fluorescent imaging light was kept at low levels as not to contribute or alter the lesion phenotype and accompanying immune response. Therefore, imaging two fluorescent channels to simultaneously view neutrophils and microglia in the same animal would have required at least 2X the visible light exposure for imaging. The imaging light levels used in the current study were carefully examined in our previous publications as to not create additional light damage (Joseph et al 2021).
(3) Along the same lines as above, the phase contrast ONL images at time points from 3-day to 2-month post laser injury are not shown and the absence of this data is not addressed. This missing data pertains only to the in vivo imaging mice model but are conducted in histology that adequately conveys the time-course of cell loss in the ONL.
The ocular preparation of the phase contrast data in figure 2, unfortunately developed an anesthesia induced cataract that precluded adequate image quality. This is not uncommon in long-term mouse ocular imaging preparations (Feng et al 2023). Instead, we chose to include the phase-contrast data to show the visually compelling intact and disrupted ONL damage for baseline and 1 day to show that the damage is not only focal, but also shows clear disruption to the somatic layers of the photoreceptors.
It is suggested that the reason be elaborated for the exclusion of this data and the simultaneous imaging of microglia and neutrophils mentioned above.
We agree and we have included the reason for the “not acquired” data within the figure 2 legend:
“Phase contrast data was not acquired for time points 3 days-2 months due to development of cataract which obscured the phase contrast signal”
Also, it would be valuable to further qualify and check the claims in the Discussion that "ex vivo analysis confirms in vivo findings" and "Microglial/neutrophil discrimination using label-free phase contrast"
We maintain that ex vivo analysis both corroborates and in many cases, confirms our in vivo findings. We feel this is a strength of our manuscript rather than a qualifier. A) Damage localization is visible with OCT and confocal/phase contrast AOSLO in a region that matches the DAPI loss we see ex vivo. B) Disruption of the ONL seen with in vivo AOSLO is of the same size, shape and location as the ONL damage quantified ex vivo. C) No damage or disruption was seen in locations above the lesion with OCT or AOSLO, which matches our finding that only the ONL shows loss of nuclei whereas other more superficial layers are spared. D) Microglial localization is found both in vivo and ex vivo and E) lack of neutrophil aggregation or extravasation was neither seen in vivo or ex vivo. Given the evidence above, we contend that this strong synergistic and complementary approach corroborates the experimental data in two ways of studying this tissue.
We agree that the claims made in the section entitled “Microglial/neutrophil discrimination using label-free phase contrast” are not strongly supported by the phase-contrast imaging presented in this paper. Accordingly, we have since removed this section based on reviewer suggestion.
Recommendations for the authors:
Reviewer #1 (Recommendations for the authors):
(1) Based on the title and abstract, the main focus of the manuscript appears to be the immune response. However, most of the manuscript is dedicated to the authors' imaging technique. Additionally, several important concerns regarding the investigation of the immune response in the retina need to be addressed.
We understand that emphasis may appear to be on the imaging technique, however, because AOSLO is not a widely used technology, we are committed to explaining the technique so that it both builds awareness and confidence in the way this exciting new data is acquired.
(2) The authors indicate '1 day post-injury' as a timeframe spanning between 18 and 28 hours post-injury. This is a rather wide window of time, which could potentially affect the analysis. It is necessary to demonstrate that there is no significant difference in the immune response, particularly in terms of microglial morphology and branch orientation, between 18 and 28 hours post-injury.
We agree that a fine time scale may show even greater insight to the natural history of the inflammatory response. However, we feel that our chosen time points go above and beyond the temporal precision that is offered by other investigations, especially considering the novel multi-modal imaging performed here. Studies using finer temporal sampling are poised for future investigation.
(3) The authors should consider using additional markers or complementary techniques to differentiate between microglia and recruited macrophages, such as incorporating immunohistochemistry with P2RY12, a specific marker for microglia that helps distinguish them from macrophages, and CD68 or F4/80, markers for recruited macrophages. It is also crucial for the authors to include a discussion addressing the limitations of using Cx3cr1GFP mice and the potential impact on result interpretation. It is fundamental to validate the findings and clarify the roles of microglia and macrophages.
The wonders of current IHC is that there are myriad antibodies and labels that “could” be used. We used what we felt were the most compelling for this stage of early investigation. We look forward to studies that employ this wider range of labels. See our response to reviewer 1’s first comment above for addressing the limitations of using Cx3CR1 mice.
(4) Analyzing neutrophil responses at 24 hours post-injury may be too late to capture the critical early dynamics of inflammation. By this time, the initial recruitment and activation phases of neutrophils may have already peaked or begun to resolve, potentially missing key insights into the immediate immune response. The authors should conduct additional analysis of neutrophil responses at earlier time points post-injury, such as 6 or 12 hours. Including these time points would provide a more comprehensive and conclusive analysis of the neutrophil response, helping to delineate the progression of inflammation and its implications for subsequent healing processes.
This point has been addressed above. Briefly, we have now included a new experiment (and figure + video) that shows no neutrophil extravasation at earlier time points. We thank the reviewer for this helpful suggestion.
Reviewer #2 (Recommendations for the authors):
This paper is extremely long, and in the perspective of this reviewer, needs to be better organized.
(1) There was a lengthy description and verification of light-induced injury and longitudinal tracking of healing, which I believe can be further cleaned up and made more succinct.
We have cleaned-up and re-organized the manuscript (see above response for details). Manuscript has been reorganized and reduced by 8%.
(2) The intention/goal of the paper can be further strengthened. On page 33: "to what extent do neutrophils respond to acute neural loss in the retina?" This particular statement is so clear and really brings out the purpose of this study, and it will be great to see something like this in the opening statement.
We thank the reviewer for this excellent suggestion. We have modified the final paragraph of the introduction to strengthen our study’s intention.
P4 L45-47: Here, we ask the question: “To what extent do microglia/neutrophils respond to acute neural loss in the retina?” To begin unraveling the complexities in this response, we deploy a deep retinal laser ablation model.
(3) The figures are not mentioned in the manuscript in the order they were numbered. It makes it extremely challenging to follow along. The methods/results sections started with Figure 1, then on to Figure 4, then back to Figures 2 and 3, etc. This reviewer recommends re-organizing figures and their order of appearance so the contents of the figures are referred to in the paragraph in the most efficient and clear manner.
We have re-organized the appearance of figure references throughout the paper.
(4) Figure 2: phase contrast was not acquired on days 3, 7, and 2 months. Please briefly explain the reason in the caption.
Addressed above.
(5) Figure 4 OPL layer, the area highlighted in a dashed circle was meant to demonstrate that perfusion was intact, but I cannot see the flow in the highlighted area very well at day 7 and 2 months (especially 2 months). Please explain.
Perfusion maps are often difficult to interpret as a static image. Therefore, we have additionally provided the raw video data (“OPL_vasculature_7d” and “OPL_vasculature_2mo”) which helps visualize active perfusion. To the reviewer’s point, videos reveal that RBC motion is maintained in the capillaries of this location.
(6) While there's a thorough discussion of the biological impact of the finding, the uniqueness of the imaging technique can be better highlighted. Immune response toward injury is highly dynamic and is often the first step of wound healing. To observe such dynamic events longitudinally in the living eye at the cellular level, it requires a special imaging technique such as the type addressed here. The author can better address the technical uniqueness of studying this type of biological event for readers less familiar with AOSLO.
We agree and following the reviewer’s suggestion have further emphasized the advance in the current manuscript in two additional places:
(1) Within the introduction
P3-4 L21-42: “A missed window of interaction is highly problematic in histological study where a single time point reveals a snapshot of the temporally complex immune response, which changes dynamically over time. Here, we use in vivo imaging to overcome these constraints.
Documenting immune cell interactions in the retina over time has been challenged by insufficient resolution and contrast to visualize single cells in the living eye. The microscopic size of immune cells requires exceptional resolution for detection. Recently, advances in AOSLO imaging have provided micron-level resolution and enhanced contrast for imaging individual immune cells in the retina and without requiring extrinsic dyes(7,23). AOSLO provides multi-modal information from confocal reflectance, phase-contrast and fluorescence modalities, which can reveal a variety of cell types simultaneously in the living eye. Here, we used confocal AOSLO to track changes in reflectance at cellular scale. Phase-contrast AOSLO provides detail on highly translucent retinal structures such as vascular wall, single blood cells(27–29), PR somata(30), and is well-suited to image resident and systemic immune cells.(7,23) Fluorescence AOSLO provides the ability to study fluorescently-labeled cells(25,31,32) and exogenous dyes(27,33) throughout the living retina. These modalities used in combination have recently provided detailed images of the retinal response to a model of human uveitis.(23,34) Together, these innovations now provide a platform to visualize, for the first time, the dynamic interplay between many immune cell types, each with a unique role in tissue inflammation.”
(2) Within the discussion
P34-35 L656-662 “Beyond the context of this specific finding, we share this work with the excitement that AOSLO cellular level imaging may reveal the interaction of multiple immune cell types in the living retina. By using fluorophores associated with specific immune cell populations, the complex dynamics that orchestrate the immune response may be examined in this specialized tissue. This work and future studies may reveal further insights to the interactions of single immune cells in the living body in a non-invasive way.”
Reviewer #3 (Recommendations for the authors):
Some other comments:
(1) The reader may wonder why if all findings are confirmed by histology would an in vivo imaging model be needed. This does not need a generalized explanation given the typical virtues of an in vivo model, but perhaps the authors may want to amplify their findings in the current context, for example, those on the shorter minutes to hours timescales (Figure 2, Supplement 1) that would have been resource and time intensive, and likely impossible, to gather via histology alone.
The reviewer appropriately underscores the utility of in vivo imaging above histological-only investigation. In response, we have added text in the introduction to emphasize the nuanced, but important value of both longitudinal imaging as well as dynamic imaging which is not possible with conventional histology (e.g. blood perfusion status, immune cell interactions etc.)
P3-4 L21-42 (these points also addressed in response to reviewer #2 above)
(2) A few questions and comments on the laser ablation model
- It is alluded to in the Discussion in Lines 519-521 that the procedure is highly reproducible (95%) but the associated data for this repeatability metric is not shown.
We agree that the criterion for determining a “successful lesion” requires further elaboration. Therefore, we have now included the criteria for successful lesions in the methods as well as discussion (in bullet below):
Methods:
P9-10 L129-133: “This protocol produced a hyper-reflective phenotype in the >40 locations across 28 mice. In rare cases, the exposure yielded no hyper-reflective lesion and were often in mice with high retinal motion, where the light dosage was spread over a larger retinal area. These locations were not included in the in-vivo or histological analysis.”
- The methods state that a 24 x 1-micron line is focused on the retina, but all lesions seem to appear elliptical where the major to minor axis ratio is a lot smaller than this intended size. One wonders what leads to this discrepancy.
We expect that this observation is related to the response above, we have added the following:
Discussion:
P27 L497-505: “The damage took on an elliptical form, likely due to: 1) Eye motion from respiration and heart rate which spreads the light over a larger integrative area (rather than line). 2) The impact of focal light scatter. 3) A micron-thin line imparting damage on cells that are many microns across manifesting as an ellipse. The majority of light exposures produced lesions of this elliptical shape. In a few conditions, for the reasons described above, the exposure failed to produce a strong, focal damage phenotype. To improve lesion reproducibility, future experiments should control for subtle eye motion affecting light damage, especially for long exposures.”
(3) Lastly, a thickening is noted in the ONL after laser injury that seems to cause a thinning of the INL as well (Figure 3) which may increase the apparent INL nuclei density.
The reviewer’s careful eye finds local swelling after injury. However, despite swelling, the segregation between INL and ONL was maintained in all days we examined. Thus, no ONL cells were included in INL counts (see figure 3A & 3D).
Also, the ONL - inner (panel B) seems to show a little reduction in cell density in the same elliptical shape as the outer ONL in panel C.
We agree with this observation and was one of the reasons we included this detailed analysis of both the inner and outer half of the ONL. Our finding is that there is more prominent loss of nuclei in the outer half of the ONL. While the mechanism for this is not understood, we felt it was an important finding to include and further shows the axial specificity of the light damage we are inducing (especially at day 1 observation).
Lastly, the reduction in nuclear density is visually obvious in the ONL at the 1 and 3-day time points but the p-statistic does not seem to convey this. One may consider performing the analysis on panel F on a smaller region surrounding the lesion to more reliably reveal these effects.
Related to the response above, the ONL shows a persistence of nuclei in the upper half of that layer, whereas the outer half, shows a visible reduction. Therefore, we expect that the reviewer is correct that a statistical analysis that considers just the outer half of the ONL would likely show a strong statistical significance. The challenge, however, is that our analysis strategy counted all cells within a 50 micron diameter cylinder through the entirety of the ONL (meaning strong loss in the outer half was attenuated by weak loss in the inner half). A more detailed sub-layer analysis is challenging given the notable retinal remodeling over days-to-weeks that make it challenging to attribute layers within the ONL as viable landmarks for the requested analysis.
(4) In Figure 6, the NIR confocal image and fluorescent microglia seem to share the same shape, starting from the OPL and posterior to it. This is particularly evident in the 3 and 7-day time points in the ONL and ONL/IS images. This departs from lines 567-577 where the claim is made that the hyperreflective phenotype in NIR images does not emerge from the microglia and neutrophils. This discrepancy should be clarified. It may be so that the hyperreflective phenotype as observed by Figure 2 at shorter timescales is not related to the microglia but the locus of hyper-reflections changes at longer time scales to involve the microglia as well as in Figure 6. One potential clue/speculation of the common shapes/size in confocal hyper-reflectance and fluorescent microglia of Figure 6 comes from Figure 9 where the microglia seem to engulf the photoreceptor phagosomes in the DAPI stains. It is possible that the hyper-reflections arise from the phagosomes but their co-localization with microglia seems to demonstrate a shared size/shape. As an addendum to the first point, such correlations are a power of the in vivo model and impossible to achieve in histology.
The reviewer shows a deep understanding of our data. We agree with many of the points, but for the purpose of the paper many of the above offerings are speculative and we have chosen not to elaborate on these points as it is not definitive from the data. Instead, we direct the reader to an important finding that within hours, the hyper-reflective phenotype is seen in both OCT and AOSLO, whereas microglial somas/processes have not yet migrated into the hyper-reflective region. We have now emphasized this point in the discussion section:
P29-30 L543-552: “A common speculation is that the increased backscatter may arise from local inflammatory cells that activate or move into the damage location. In our data, confocal AOSLO and OCT revealed a hyperreflective band at the OPL and ONL after 488 nm light exposure (Figure 2a, b). We found that the hyperreflective bands appeared within 30 minutes after the laser injury, preceding any detectable microglial migration toward the damage location (Figure 2 – figure supplement 1 and Figure 6 – figure supplement 1). We thus conclude that the initial hyperreflective phenotype is not caused by microglial cell activity or aggregation.”
