Extrinsic Regulation of Optic Nerve Axon Regeneration in the Adult Central Nervous System

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript (cells-4416855) entitled  “Extrinsic Regulation of Optic Nerve Axon Regeneration in the Adult Central Nervous System”, by A. Adhikary and co-workers is a comprehensive and timely review addressing the role of the extrinsic environment in adult optic nerve axon regeneration. The manuscript covers an extensive body of literature and discusses many of the principal extracellular factors known to influence retinal ganglion cell (RGC) regeneration, including extracellular matrix remodeling, inhibitory guidance molecules, inflammatory responses, metabolic alterations, disease-specific microenvironments, and emerging therapeutic strategies.

Overall, the review is well organized, clearly written, and supported by an extensive bibliography. The graphical figures are of excellent quality and greatly facilitate understanding of complex biological processes. The topic is highly relevant, particularly given the increasing recognition that successful optic nerve regeneration depends not only on enhancing intrinsic neuronal growth programs but also on modifying the surrounding tissue environment.

I believe that this manuscript has the potential to become a valuable reference in the field. The following comments are intended to strengthen its conceptual impact and further improve its scientific value.

1. Major comments

1.- Strengthen the conceptual framework of the review. The principal limitation of the manuscript is that, although highly comprehensive, it remains predominantly descriptive. Most sections summarize the available literature on individual extracellular factors, but the review would benefit from a stronger conceptual synthesis. Rather than presenting extracellular matrix remodeling, inflammation, guidance molecules, metabolic alterations and vascular changes as relatively independent processes, I encourage the authors to integrate these mechanisms into a unified biological model. The concept of a continuously evolving regenerative microenvironment or dynamic regenerative niche could provide an excellent framework through which these different processes interact spatially and temporally after injury. Such an integrative perspective would substantially increase the originality and impact of the review.

2.- Emphasize temporal dynamics. One of the most interesting aspects introduced by the authors is the temporal evolution of the extrinsic environment. However, this concept deserves greater development throughout the manuscript. Several extracellular signals are initially beneficial but later become inhibitory. Similarly, inflammatory responses, extracellular matrix remodeling, glial activation and metabolic changes evolve continuously during regeneration. A dedicated section or conceptual figure summarizing the temporal sequence of these events would greatly strengthen the manuscript and provide readers with a clearer mechanistic understanding.

3.- Expand the discussion of interactions among extrinsic factors. Many components discussed in separate sections are, in reality, highly interconnected. Highlighting these reciprocal interactions would provide a much more integrated view of the regenerative process.

4.- Improve the discussion of mechanobiology. The manuscript briefly discusses mechanosensitive pathways, particularly Piezo1, but this section could be considerably expanded. Recent evidence suggests that mechanical properties of the extracellular matrix are not merely passive barriers but active regulators of cellular behaviors. Matrix stiffness, mechanotransduction, cytoskeletal remodeling and glial responses are increasingly recognized as central regulators of regeneration. A broader discussion of mechanobiology would provide an important emerging perspective that aligns well with the scope of Cells.

5.- Further develop translational implications. The section describing therapeutic strategies is informative but could benefit from a more critical analysis. Instead of presenting individual therapeutic approaches sequentially, it would be useful to discuss why many successful experimental interventions have failed to translate into clinical practice. The authors may wish to emphasize that regeneration will probably require combinatorial approaches targeting multiple components of the injury microenvironment simultaneously and at appropriate temporal windows rather than single molecular targets.

6.- Discuss the relationship between intrinsic and extrinsic mechanisms. Although the review intentionally focuses on extrinsic regulation, regeneration ultimately depends on continuous interactions between intrinsic neuronal growth competence and the extracellular environment. Including a brief section summarizing how intrinsic and extrinsic mechanisms cooperate (or occasionally counteract each other) would provide a more balanced and biologically complete perspective.

7.- Future perspectives. The final section would benefit from moving beyond listing promising therapeutic targets. Instead, the authors could propose future research priorities, including integration of multi-omics approaches, tissue engineering, etc. Such perspectives would provide readers with a clearer vision of future directions in the field.

1. Minor comments

1.- Some sections describing signaling pathways are highly detailed, whereas others are comparatively brief. Improving the balance between sections would enhance readability.

2.- The figures are excellent and represent one of the major strengths of the manuscript. Nevertheless, an additional integrative figure summarizing the temporal evolution of the regenerative microenvironment—from acute injury through chronic remodeling and potential regeneration—would further increase the educational value of the review.

3.- Tables 1–3 contain valuable information. Including a final summary table integrating extracellular barriers, their molecular mediators, downstream signaling pathways, and potential therapeutic interventions would provide readers with an excellent overview.

4.- The conclusion could be strengthened by emphasizing that the extrinsic environment should not be viewed simply as a collection of inhibitory factors but rather as a highly dynamic and biologically regulated system whose composition changes continuously during degeneration and regeneration.

Overall, this manuscript addresses an important and rapidly evolving area of neurobiology. It is comprehensive, well documented and supported by excellent illustrations. The suggested revisions are intended primarily to increase its conceptual integration and translational significance rather than to modify its overall structure. Addressing these points would substantially enhance the originality, scientific depth and long-term impact of this review.

Author Response

Major: We thank the reviewer for this suggestion. We have reframed the manuscript around the concept of a dynamic regenerative niche, introduced explicitly in the abstract and introduction and carried through the Synthesis section (10.1). Rather than presenting vascular, immune, glial, matrix, and metabolic factors as independent barriers, we now explicitly state that these compartments are spatially co-localized and temporally coordinated, and we demonstrate this through specific examples throughout the manuscript (e.g., Section 5.2, where inflammation-driven astrocyte activation is shown to drive the CSPG deposition described in Section 3.1). 

We thank the reviewer for this suggestion. We have integrated the bridges between each phase post-injury, so it creates evolving temporal dynamics rather than independent stages. Rather than summarizing states, a clear description of the processes was written along with a figure for deeper understanding. We have also added a new integrative figure (Figure 5) that consolidates the temporal evolution of the extrinsic injury microenvironment across vascular, axonal, innate immune, astrocytic, extracellular matrix, and metabolic compartments on a single timeline spanning acute injury (0 to 24h) through chronic remodeling (21+ days). RGC survival is plotted beneath the mechanistic tracks, including subtype-specific vulnerability of α-RGCs versus ipRGCs, allowing readers to align cellular and molecular events with functional outcome. 

We agree that the reciprocal interactions among extrinsic factors deserved more explicit treatment. The new integrative figure (Figure 5) includes directional arrows connecting compartments to indicate causal signaling, including BBB disruption permitting leukocyte infiltration, debris-driven innate immune activation, M1 microglia-driven astrocyte neurotoxic differentiation, and reactive astrocyte-driven ECM deposition. Critically, the figure also depicts a feedback loop by which chronic CSPG accumulation in the extracellular matrix re-inhibits axon regrowth, illustrated at the point of growth cone collapse, showing that these processes form a continuously interacting system rather than a linear cascade.

Section 3.1 now includes an expanded discussion of mechanobiology, addressing substrate stiffness and mechanical gradient effects on axon pathfinding, and detailing how Piezo1 activation suppresses axon regeneration through calcium-dependent signaling. We also discuss Piezo1's role in glial cells, where it regulates astrocyte proliferation in the optic nerve head, and its potential as a therapeutic target given its accessible plasma membrane localization. 

We thank the reviewers for this suggestion. Section 9 now opens by noting that, although organized by mechanism for clarity, single-node interventions often have limited clinical impact because the optic nerve microenvironment is highly interconnected. We illustrate this directly in Section 9.2: complete knockout of the CSPG receptor PTPσ reduces inhibitory signaling but produces axon misdirection errors in regenerating sciatic nerve, demonstrating that removing one inhibitory node can disrupt the broader guidance information an axon depends on. We conclude Section 9 with a new subsection (9.5, Toward Combinatorial, Time-Sensitive Interventions) explicitly addressing why current therapies remain largely theoretical and why future approaches will likely require combinatorial, timing-aware strategies rather than single molecular targets. 

Section 10.2 now includes a dedicated discussion of the interplay between intrinsic and extrinsic mechanisms, noting that extracellular cues can modify intracellular signaling and vice versa, such that an inhibitory extrinsic environment can limit regenerative capacity even when intrinsic growth pathways are pharmacologically upregulated. Because the primary goal of this review is the extrinsic environment, we kept this discussion concise while still directly addressing how the two mechanisms interact.  

We thank the reviewer for this suggestion. Section 10.4 (Future Methodological Directions) now explicitly discusses multi-omics approaches, including transcriptomic, proteomic, and lipidomic integration, and continued development of ECM-based tissue engineering, including bioscaffolds and synthetic conduits, as priorities for future research.

 We thank the reviewer for this suggestion, which we agree substantially strengthens the manuscript. We have added Figure 5, an integrative temporal figure summarizing the evolution of the regenerative microenvironment from acute injury through chronic remodeling, including cross-compartment causal arrows, cellular-level illustrations at key transition points in each compartment, and RGC survival trends across the injury timeline. 

 We thank the reviewer for this suggestion. Revisions have been made to make each section as detailed as others. 

 

Reviewer 2 Report

Comments and Suggestions for Authors

It is a pleasure to read this review about extrinsic regulation of optic nerve axon regeneration in the adult central nervous system. The design, the manuscript text and outline is fine - only minor things like spacing in line 585 are to correct.

To complete the ample presentation of factors a major suggestion is: please also mention guiding cues by electrode stimulation either from outside or integrated in scaffolding. Because some studies have shown that electrical stimulation improves survival of transected retinal ganglion cells in rats (Morimoto et al. 2005), that it delays degeneration and improves survival and function of photoreceptors (Zhou et al. 2012) and exhibits neuroprotective effects in  retinas of rats (Schatz et al. 2012, Ciavatta et al. 2013).

 

Author Response

We thank the reviewer. The revisions and formatting have been corrected, fixing minor errors.

The paragraph in Section 9.1 was revised to include electrical stimulation as an emerging regenerative therapy, citing the specific studies raised by the reviewer (Morimoto et al. 2005, Zhou et al. 2012, Schatz et al. 2012, Ciavatta et al. 2013), and now also discusses the neurotrophic mechanism (BDNF and IGF-1 release from Müller cells) underlying these effects, as well as more recent work on electrical field-mediated long-distance axon regeneration and conductive biomaterial scaffolds.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Thank you for your careful revision of the manuscript entitled “Extrinsic Regulation of Optic Nerve Axon Regeneration in the Adult Central Nervous System.”

The revised version has satisfactorily addressed the main concerns raised during the previous review. The manuscript is now clearer, better integrated, and provides a more balanced discussion of the dynamic and interconnected extrinsic factors influencing optic nerve axon regeneration.

I have no further substantive scientific comments and consider the manuscript suitable for publication. Any remaining minor typographical, formatting, or reference inconsistencies can be addressed during final editorial preparation.