Improved Locomotor Recovery in a Rat Model of Spinal Cord Injury by BioLuminescent-OptoGenetic (BL-OG) Stimulation with an Enhanced Luminopsin

Round 1

Reviewer 1 Report

The manuscript by Ikefuama et al. presents the application  of a very  interesting bioluminesnce-based optogenetic tool (BLOG) on a rat model of spinal cord injury. The approach, relying on  an light-activated ion channel with improved ion movimentation capability, allows for a non-invasive delivery of the activating substrate. This,  with respect to the current state-of-the -art, is a interesting point of novelty. Data on locomotor performance show that the approach is effective to a certain extent.

Said that, I would suggest a few points to the authors to render solid the paper scientific soundness.

1. As far as I got to understand, this version of BLOG with Cheriff opsin is presented here for the first time. Eventhough, on a line of principle this should increase the efficacy of the method, no data are provided with a side-by-side electrophysiological comparison (current amplitude vs CTZ) with previously published tools. If this is out of the lab expertise range, BBB scores and subscores could be measured in animals expressing previous constructs with the same CTZ delivery protocol. 

2. following up on the previous point, one might wonder what is more effective and the actual difference in the effect size resulting from the different delivery protocol. This is fundamental.    

3. The designed method according  to the author view support the manuscript main message about non-invasiveness (see title).  It is fine of course ti consider peripheral delivery non-invasive but one cannot forget the fact tha such constructs are expressed by means of viral vectors. While in the most of the text the authors make this point clear, this is not the case in some other places (title). This could result misleading. 

Author Response

1. We have included patchclamp measurement of LMO3 vs LMO3.2 for direct comparison as was done for characterization of other LMO constructs to show improved amplitude of response to CTZ.

2. Different delivery protocols and dosing timelines could certainly impact recovery outcomes and is something we are actively pursuing for follow up experiments but is outside the scope of the current study as we only sought to determine if LMO3.2 could provide positive results when using a lower does of CTZ than would be effective for the prior construct.

3. We updated the title to drop the phrasing “non-invasive”

Reviewer 2 Report

In their study, Ikefuama and coll. have tested a new generation of Non-Invasive BioLuminescent-OptoGenetic (BL-OG) Stimulation to induce neuronal activation and improves hindlimb motor function following a severe thoracic contusion SCI. This stimulation can only occur when luciferin coelenterazine (CTZ) is present. They had previously shown that such an approach can be efficient when CTZ was applied through a lateral ventricle cannula, and in the present study, they tested a new generation of LMOs based on opsins with higher light sensitivity which would allow for peripheral delivery of the CTZ. This is a potential innovative avenue of treatment that seems promising and needs a number of proof-of-concept studies like the present one. The manuscript reads well and the methods and results are correctly described although a little bit succinct. The statistical analysis looks fine. I hope the following comments can help to improve the manuscript

Major comments:

- It is not clear when the viral injection occurs? Is it before injury as previously described in ref 24? This should be clarified in the methods. If so, this is an important limitation to the study for translational approach that needs to be discussed in a dedicated paragraph. On the contrary, could AVV-hSyn-HB9-LMO injection be done after injury? If so, what impact would have virus injection after injury?

- Regarding control groups, what about animals that did not express LMO3.2? How many were they? Why? And what is the effect of previous injection on functional performance after surgery? It would be interesting to present a control group for both viral injection and CTZ treatment. Please provide BBB score and/or catwalk results that are expected after surgery but without any intervention.

- Why is viral injection at T13-L1 and injury done at T9? What is the rationale?

- Introduction needs to provide more details on previous studies on neural activation using BioLuminescent-OptoGenetics. What is the rationale for “neural activation” and what kind of neural activation is expected here?

Minor comments

-Too many abbreviations in the manuscript, the authors should try to reduce the number of abbreviations at least in both introduction and conclusion

- Line 158, check for repetitions in the sentence.

- Figure 5: annotations cannot be read in the current version of the figure. Please provide an improved version.

- Mat & Methods: a schematic of the experimental model is missing. Readers should not need to read the previous work to understand what has been done in the present study. Please clarify the setup and experiment setting of the present study with a figure.

Author Response

1. 

Viral injections were performed before the injury and this has been clarified in the method section with the timeline from the graphical abstract added to the main text. We used a rodent model of SCI and we wanted to see the role of immediate treatment or intervention on locomotor recovery. To achieve this aim, after the injections, we waited three weeks for viral expression before the contusion spinal cord injury. Of course when applying this approach to patients there are a variety of other genetic delivery methods that can provide rapid expression compared to AAV allowing for early time point interventions. Another approach would be peripheral injections of an AAV capable of retrograde transductions, however for the sake of experimental consistency at this time direct injections AAV is a good method for early phase rodent experiments.

2. 

In this current study, all animals expressed the LMO3.2 construct. Adding additional groups for a virus control with CTZ and virus control with vehicle would be an unnecessary duplication of previous animal studies. Please see Figure 3, sbGluc-B7+ CTZ and sbGluc-B7 + veh [24]) The viral construct used for these control groups was the luciferase tethered to the membrane without an ion channel. The results from these control groups are also consistent with spontaneous recovery for the rat model as in the work from Basso and colleagues (https://doi.org/10.1006/exnr.1996.0098). Unfortunately we did not have the CatWalk at the time of our prior experiment with the additional control groups and are unable to make a comparison.

3. 

The aim of this study is to stimulate neurons that are below the injury site and involved in locomotion. The lumbar enlargement contains the interneurons involved in hind limb central pattern generators and the motor neurons responsible for motor output; hence, the reason why we performed viral injection at T13-L1 and injury done at T9 in the thoracic region.

4. We have added more detail on the specifics of how optogenetics activate neurons in the introduction.

We have also implemented all suggested minor changes. 

Round 2

Reviewer 2 Report

I am satisfied with the authors answers. I however suggest that they provide this information in study limitations (2 sentences).