Photoreceptor loss does not recruit neutrophils despite strong microglial activation

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.

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.

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.

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.

Weaknesses:

This paper is extremely long, and in the perspective of this reviewer, needs to be better organized.

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.

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.

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.

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.

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.

(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.

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.

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.

(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.

(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. It is suggested that the reason be elaborated for the exclusion of this data and the simultaneous imaging of microglia and neutrophils mentioned above. 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"