Cold protection allows local cryotherapy in a clinical-relevant model of traumatic optic neuropathy

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1. 

   Version published to 10.7554/elife.75070 on eLife

   Mar 30, 2022
2. 

   eLife

   Mar 6, 2022

   Author Response:

   Reviewer #2 (Public Review):

   Zhang et al. describe a new method for inducing traumatic optic neuropathy in larger mammalian models that offers the additional advantage of allowing rapid administration of local therapies to the site of optic nerve injury. Furthermore, the authors build on their prior work which has demonstrated a neuroprotective effect of hypothermia provided that protease inhibitors are employed to protect against cold-induced microtubule damage. In the present manuscript, they show that an endonasal approach to accessing the optic nerve within the optic canal can be performed safely in goat without inducing optic nerve damage. They then demonstrate that experimental crush of the optic nerve within the optic canal results in progressive degeneration of retinal ganglion cell (RGC) neurons and of their …

   Author Response:

   Reviewer #2 (Public Review):

   Zhang et al. describe a new method for inducing traumatic optic neuropathy in larger mammalian models that offers the additional advantage of allowing rapid administration of local therapies to the site of optic nerve injury. Furthermore, the authors build on their prior work which has demonstrated a neuroprotective effect of hypothermia provided that protease inhibitors are employed to protect against cold-induced microtubule damage. In the present manuscript, they show that an endonasal approach to accessing the optic nerve within the optic canal can be performed safely in goat without inducing optic nerve damage. They then demonstrate that experimental crush of the optic nerve within the optic canal results in progressive degeneration of retinal ganglion cell (RGC) neurons and of their axons which form the optic nerve; this occurs over a period of several months, a similar time course to traumatic optic neuropathy in humans. Transcriptional profiling of mRNA obtained from the optic nerve at its site of injury identified changes in gene expression related to molecular pathways involved in inflammation, ischemia, and cellular metabolism. The authors then proceed to apply local hypothermia or protease inhibitor administration (individually or in combination) to the site of optic nerve crush for two minutes and observed a decrease in axonal degeneration in the subsequent months, although without an improvement of conduction of visual information by the affected optic nerve. Finally, the authors describe a computer program which uses computed tomography scans to evaluate thousands of potential endonasal approaches to access the prechiasmal optic nerve in large and medium sized mammals, and they proceed to successfully perform optic nerve exposure and experimental crush in a macaque model.

   The authors' interpretation of the data is generally accurate; however, their conclusions about the impact of the work are somewhat overstated.

   We appreciate the reviewer’s insightful and precise comments, which definitely help improve our manuscript. We have scaled back the conclusions about the impact of our work, and revised our manuscript point by point according to the reviewer’s comments.

   Strengths:

   The prevailing use of rodent models of traumatic optic neuropathy in the field is problematic, and the authors' efforts to use larger mammalian models may be helpful in understanding the pathophysiology of and developing treatments for traumatic optic neuropathy in humans. The computer program that evaluates and recommends detailed surgical approaches and instrumentation is novel and would be quite useful to other investigators attempting to perform similar endonasal procedures. The authors make use of multimodal assessments of the goats and macaques [e.g. quantitative retina and optic nerve histology; optical coherence tomography measurements of retinal layers; pupillary light reflex assessment; and electrophysiology studies including visual evoked potential (VEP) and pattern electroretinography (PERG)] to convincingly demonstrate that the endonasal procedure itself can be performed without inducing progressive optic nerve damage and that experimental optic nerve crush using this procedure induces the expected profound decrease in optic nerve function, associated with progressive degeneration of RGC cell bodies and axons. The histological assessment of goat optic nerves following the local hypothermia/protease inhibitor treatment demonstrates a convincing reduction in the absolute number of RGC axons that are lost at 1 month after optic nerve crush.

   Thank you very much.

   Weaknesses:

   The premise that optic nerve crush within the optic canal is much more physiologically relevant than other existing animal models is overstated: while the optic canal is believed to be the most common site of injury to the optic nerve, most human cases of traumatic optic neuropathy occur as a result of indirect mechanisms rather than compression/crush-namely, stretching and shearing forces are applied to the optic nerve where it is tethered to the periosteum of the optic canal by its dura. The authors' example of a bony fragment compressing the optic nerve within the optic canal (Figure 1B) is relatively rare and would actually represent one of the few cases where a surgical intervention (to relieve acute optic nerve compression) might be considered clinically justifiable.

   We agree with the reviewer that “most human cases of traumatic optic neuropathy occur as a result of indirect mechanisms rather than compression/crush”. To address the reviewer’s concern, we updated our discussion as: “Recently, rodent models have been developed using indirect mechanisms (apply periorbital ultrasound or skull weight drop) to induce distal ON injury. Compared with direct optic nerve compression or crush, these models are likely more clinically relevant since most clinical TON cases are indirect and due to force transmission (39-41). However, due to force scattering, unwanted and uncontrolled collateral damage to the eyeball, contralateral optic nerve, orbit or skull often occur in these models (39,40). Additionally, the success rate of these modeling methods is not as high as direct optic nerve crush; for example, 10% mice died immediately after head weight drop (39). Moreover, extension of these modelling methods to large animal species has not been reported. Therefore, clinically translatable, local treatment of injured ONs via trans-nasal endoscopy cannot be performed in these small animal models.”

   To further address the reviewer’s concern, we have added the following statement to the “Limitations of this study” section in the revised manuscript: “Our TON model is clinically relevant in terms of injury site, subsequent spatiotemporal pattern of retrograde axonal degeneration, and availability of trans-nasal local treatment. However, the mechanism of optic nerve injury in our model differs from that in most clinical TON cases, in which the intra-canalicular optic nerve is injured by an indirect mechanism (stretching and shearing forces), rather than by direct compressing forces.”.

   We respectfully disagree that “a bony fragment compressing the optic nerve within the optic canal (Figure 1B) is relatively rare”, at least in China. According to our previous clinical study of 1275 patients with indirect traumatic optic neuropathy (PMID: 27267448), bony fracture of the optic canal is not rare: 50% of patients had a visible optic canal fracture on high-resolution CT scans, and an additional 20% had a visible optic canal fracture under trans-nasal endoscopy (because endoscopy provides excellent illumination and a magnified view of the optic canal).

   The transcriptomic profiling at three locations along the visual pathway in post-trauma goats only showed differences in expression at the location of optic nerve injury, and not within the retina or proximal optic nerve on the affected side. The authors assert that the high rate of expression changes in pathways relevant to ischemia, inflammation and metabolism indicates "that targeting these pathways with local treatment could alleviate secondary damage." This is overstated. While such profiling may be useful for hypothesis generation, it was not followed up by any experiments to determine whether these expression changes are actually detrimental to the optic nerve. Some of them may very well be compensatory, such that inhibiting them may only exacerbate damage. Furthermore, given that these transcriptional changes are not seen in the retina (where RGC nuclei reside) or in the proximal optic nerve, this would suggest that the observed transcriptional changes at the site of injury are actually occurring in non-neuronal cells (e.g. astrocytes, oligodendrocytes, microglia). The authors should convey that the changes they observe are unlikely intrinsic to the optic nerve axons.

   We appreciate the reviewer’s insightful explanation and agree that our original claim is premature. We deleted the statement " targeting these pathways with local treatment could alleviate secondary damage" in the Results section. Additionally, we have replaced the previous sentence in the Discussion, which stated: " Instead, we targeted hypothermia to the injured pre-chiasmatic ON to prevent early changes in ischemia, inflammation, and metabolism transcripts (as revealed by RNA-sequencing at 1 dpi)." with “Instead, we targeted hypothermia to the injured pre-chiasmatic ON according to early transcriptomic changes in ischemia, inflammation, and metabolism pathways.”.

   We also agree with the reviewer’s comment that these transcriptomic changes at the injured optic nerve mainly occurred in the micro-environment of non-neuronal cells. The major advantage of our large animal model is the ability to modulate the micro-environment around the axons of the distal optic nerve, including glial cells, vasculature, connective tissues and the extracellular matrix. To incorporate the reviewer’s comment, we have added the following statement in the Results section: “These transcriptomic changes at the injury site were unlikely intrinsic to the distal optic nerve, and more likely occurred mostly in non-neuronal cells in the micro-environment.”

   The lack of any rescue of the pupillary light reflex or of visual evoked potential responses after local hypothermia/protease inhibitor treatment suggests that the physiological significance of any anatomical rescue by this treatment is minimal. If a number of axons survive but cannot conduct sufficient visual signal to stimulate the pupil response or stimulate the visual cortex, then the local treatment (even when applied immediately after trauma in this model, unlike in human patients in which a delay of a number of hours would be required at the very least) cannot be considered a substantial success. The authors also characterize the goats at 1 month post-injury, so one cannot say whether the statistically significant improvement in axon loss with the combined treatment would be durable at the later 3-month time point.

   We agree with the reviewer that the local treatment with hypothermia/protease inhibitor did not achieve functional recovery of the visual pathway, and thus it could not be considered a substantial success. Our work provides a safe and clinically translatable approach to modulate the inhibitory extrinsic environment at the injury site. Although in our current study, application of local treatment with hypothermia/protease inhibitor does not achieve eye-to-brain functional recovery, and its long-term therapeutic effect is unclear, these results are encouraging because they show some benefit of local treatment. Another important feature of our large animal model is that local treatment of the extrinsic environment at the distal optic nerve can be combined with currently available intravitreal treatments, such as gene therapy to boost intrinsic axon regrowth capacity, to target both extrinsic and intrinsic factors. To address the reviewer’s comment, we have added the following to the discussion in the “Limitations of this study” section: “Additionally, the current local treatment did not achieve functional recovery of the eye-to-brain pathway, and its long-term therapeutic effect is unclear.”

   As the authors acknowledge, the use of larger mammals prevented them from conducting studies with a large n. As a result, their analyses are underpowered.

   We agree with the reviewer that, in an ideal scenario, increasing the sample size would improve the power of the analysis. In this study, however, ethical issues and limitations of housing space and other resources, constrain the sample size of large mammals. We include this point in our “Limitations of this study” section.

   Because of the dissimilarities between their model and the most common mechanism of human traumatic optic neuropathy and because of the lack of a clinically significant rescue of the optic neuropathy when the local hypothermia/protease inhibitor treatment was applied, the authors' assertion that their model may "trigger a paradigm shift " for traumatic optic neuropathy research should be scaled back.

   We agree with the reviewer that it is too early to state that our model may “trigger a paradigm shift”. We need more effective local treatments to prove the value of this model and the novel therapeutic approach. Therefore, we have deleted “trigger a paradigm shift” in the Introduction and Discussion.

   Read the original source
3. 

   eLife

   Mar 6, 2022

   Evaluation Summary:

   The manuscript by Zhang et al. describes the combined use of a new surgical procedure and therapeutic hypothermia to deliver local therapy to the optic nerve in large mammals. In addition, the work describes a novel computer program that can optimize surgical approaches to access the optic nerve endonasally by using anatomical parameters obtained by tomography scan. The study represents a significant step forward in the use of therapeutic hypothermia in the treatment of ocular conditions and is of interest to neurobiologists studying therapeutic interventions for acute trauma to the optic nerves or to other regions of the central nervous system.

   (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the …

   Evaluation Summary:

   The manuscript by Zhang et al. describes the combined use of a new surgical procedure and therapeutic hypothermia to deliver local therapy to the optic nerve in large mammals. In addition, the work describes a novel computer program that can optimize surgical approaches to access the optic nerve endonasally by using anatomical parameters obtained by tomography scan. The study represents a significant step forward in the use of therapeutic hypothermia in the treatment of ocular conditions and is of interest to neurobiologists studying therapeutic interventions for acute trauma to the optic nerves or to other regions of the central nervous system.

   (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

   Read the original source
4. 

   eLife

   Mar 6, 2022

   Reviewer #1 (Public Review):

   The manuscript by Zhang et al. describes a new surgical procedure to access the optic nerve in large mammals to provide therapeutic hypothermia. Therapeutic hypothermia is a powerful idea to prevent degeneration of the nervous system following trauma or other insults. The present work offers a very important step forward in the applicability of such technology to hidden areas of the nervous system in large mammals, such as the optic nerve. A large amount of new technology has been employed and a computer approach is provided to guide the surgeons in the application of the technology to large animals and, eventually, to humans. But for a few suggestions, I believe the manuscript is well written and has the potential to reach a large audience.

   Read the original source
5. 

   eLife

   Mar 6, 2022

   Reviewer #2 (Public Review):

   Zhang et al. describe a new method for inducing traumatic optic neuropathy in larger mammalian models that offers the additional advantage of allowing rapid administration of local therapies to the site of optic nerve injury. Furthermore, the authors build on their prior work which has demonstrated a neuroprotective effect of hypothermia provided that protease inhibitors are employed to protect against cold-induced microtubule damage. In the present manuscript, they show that an endonasal approach to accessing the optic nerve within the optic canal can be performed safely in goat without inducing optic nerve damage. They then demonstrate that experimental crush of the optic nerve within the optic canal results in progressive degeneration of retinal ganglion cell (RGC) neurons and of their axons which form …

   Reviewer #2 (Public Review):

   Zhang et al. describe a new method for inducing traumatic optic neuropathy in larger mammalian models that offers the additional advantage of allowing rapid administration of local therapies to the site of optic nerve injury. Furthermore, the authors build on their prior work which has demonstrated a neuroprotective effect of hypothermia provided that protease inhibitors are employed to protect against cold-induced microtubule damage. In the present manuscript, they show that an endonasal approach to accessing the optic nerve within the optic canal can be performed safely in goat without inducing optic nerve damage. They then demonstrate that experimental crush of the optic nerve within the optic canal results in progressive degeneration of retinal ganglion cell (RGC) neurons and of their axons which form the optic nerve; this occurs over a period of several months, a similar time course to traumatic optic neuropathy in humans. Transcriptional profiling of mRNA obtained from the optic nerve at its site of injury identified changes in gene expression related to molecular pathways involved in inflammation, ischemia, and cellular metabolism. The authors then proceed to apply local hypothermia or protease inhibitor administration (individually or in combination) to the site of optic nerve crush for two minutes and observed a decrease in axonal degeneration in the subsequent months, although without an improvement of conduction of visual information by the affected optic nerve. Finally, the authors describe a computer program which uses computed tomography scans to evaluate thousands of potential endonasal approaches to access the prechiasmal optic nerve in large and medium sized mammals, and they proceed to successfully perform optic nerve exposure and experimental crush in a macaque model.

   The authors' interpretation of the data is generally accurate; however, their conclusions about the impact of the work are somewhat overstated.

   Strengths:

   The prevailing use of rodent models of traumatic optic neuropathy in the field is problematic, and the authors' efforts to use larger mammalian models may be helpful in understanding the pathophysiology of and developing treatments for traumatic optic neuropathy in humans. The computer program that evaluates and recommends detailed surgical approaches and instrumentation is novel and would be quite useful to other investigators attempting to perform similar endonasal procedures. The authors make use of multimodal assessments of the goats and macaques [e.g. quantitative retina and optic nerve histology; optical coherence tomography measurements of retinal layers; pupillary light reflex assessment; and electrophysiology studies including visual evoked potential (VEP) and pattern electroretinography (PERG)] to convincingly demonstrate that the endonasal procedure itself can be performed without inducing progressive optic nerve damage and that experimental optic nerve crush using this procedure induces the expected profound decrease in optic nerve function, associated with progressive degeneration of RGC cell bodies and axons. The histological assessment of goat optic nerves following the local hypothermia/protease inhibitor treatment demonstrates a convincing reduction in the absolute number of RGC axons that are lost at 1 month after optic nerve crush.

   Weaknesses:

   The premise that optic nerve crush within the optic canal is much more physiologically relevant than other existing animal models is overstated: while the optic canal is believed to be the most common site of injury to the optic nerve, most human cases of traumatic optic neuropathy occur as a result of indirect mechanisms rather than compression/crush-namely, stretching and shearing forces are applied to the optic nerve where it is tethered to the periosteum of the optic canal by its dura. The authors' example of a bony fragment compressing the optic nerve within the optic canal (Figure 1B) is relatively rare and would actually represent one of the few cases where a surgical intervention (to relieve acute optic nerve compression) might be considered clinically justifiable.

   The transcriptomic profiling at three locations along the visual pathway in post-trauma goats only showed differences in expression at the location of optic nerve injury, and not within the retina or proximal optic nerve on the affected side. The authors assert that the high rate of expression changes in pathways relevant to ischemia, inflammation and metabolism indicates "that targeting these pathways with local treatment could alleviate secondary damage." This is overstated. While such profiling may be useful for hypothesis generation, it was not followed up by any experiments to determine whether these expression changes are actually detrimental to the optic nerve. Some of them may very well be compensatory, such that inhibiting them may only exacerbate damage. Furthermore, given that these transcriptional changes are not seen in the retina (where RGC nuclei reside) or in the proximal optic nerve, this would suggest that the observed transcriptional changes at the site of injury are actually occurring in non-neuronal cells (e.g. astrocytes, oligodendrocytes, microglia). The authors should convey that the changes they observe are unlikely intrinsic to the optic nerve axons.

   The lack of any rescue of the pupillary light reflex or of visual evoked potential responses after local hypothermia/protease inhibitor treatment suggests that the physiological significance of any anatomical rescue by this treatment is minimal. If a number of axons survive but cannot conduct sufficient visual signal to stimulate the pupil response or stimulate the visual cortex, then the local treatment (even when applied immediately after trauma in this model, unlike in human patients in which a delay of a number of hours would be required at the very least) cannot be considered a substantial success. The authors also characterize the goats at 1 month post-injury, so one cannot say whether the statistically significant improvement in axon loss with the combined treatment would be durable at the later 3-month time point.

   As the authors acknowledge, the use of larger mammals prevented them from conducting studies with a large n. As a result, their analyses are underpowered.

   Because of the dissimilarities between their model and the most common mechanism of human traumatic optic neuropathy and because of the lack of a clinically significant rescue of the optic neuropathy when the local hypothermia/protease inhibitor treatment was applied, the authors' assertion that their model may "trigger a paradigm shift " for traumatic optic neuropathy research should be scaled back.

   Read the original source
6. 

   Version published to 10.1101/2021.11.07.467626v1 on bioRxiv

   Nov 8, 2021