Probiotic and Prebiotic Supplementation for Gastrointestinal Discomfort in Chronic Spinal Cord Injury (PRO-GIDSCI): A Randomized Controlled Crossover Trial Protocol

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

1. Lines 38-39: Up to 70% of individuals with a spinal ........ complication." This information needs to be specified; is this data worldwide reported or for some countries?
2. Line 100: "The study will be conducted at the Swiss Paraple......." This statement is grammatically wrong. Even this whole paragraph has grammatical errors.
3. Lines 108-109: "he anticipated the start of recruitment is in summer 2025 and the end of data collection is anticipated for summer 2027." What is meaning by this its not correct.
4. Heading; Study participants "will be" is not correct and should be revised. Followed the similar comments throughout the manuscript.
5. Lines 171-173: Outcome data are gathered at four time-points: baseline (T1), after the first eight- week supplementation phase (T2), after the four-week wash-out (T3) and after the second supplementation phase (T4)." This is not clear what supplementation has been added. Explain in detail.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Dear authors,

The work presented by Trunz J et al. seeks to evaluate the efficacy of probiotic and prebiotic consumption in SCI patients to improve GI function. The work is novel and addresses an important topic. However, some points require further clarification, and there are several major and minor issues regarding the experimental design that should be addressed.

Major points.

1. The study includes two groups, one receiving probiotics followed by a washout period and then prebiotic consumption. However, there is no control group, which would be necessary to establish a baseline and adequately compare the effectiveness of the treatments in improving GI dysfunction.
2. It is not clear why the authors opted to administer the probiotic treatment first and the prebiotic treatment afterwards. The oposite order might appear more straightforward. way around strategy is more clear. Could the authors please expand on the rationale behind this sequence?
3. Could the authors please expand on the rationale for selecting the specific cytokines panel, please?.

Minor points

1. In the exclusion criteria, it would be recommended to exclude patients who consumed prebiotic or probiotic prior to enrolling in the study.
2. In line 157, it is stated: "The prebiotic intervention involves taking a prebiotic supplement for 8 weeks". Earlier in the manuscript, a 4-week period was mentioned. Please clarify whether the treatment period will be 4 or 8 weeks.
3. Will the study include patients with a specific SCI level? Please justify this decision, as different injury levels may present distinct GI functional outcomes.

Author Response

Please see the attachement.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The suggested trial protocol, “Probiotic and Prebiotic Supplementation for Gastrointestinal Discomfort in Chronic Spinal Cord Injury (PRO-GIDSCI),” addresses an important clinical problem. The protocol is clear, and the chosen outcomes are appropriate. The crossover design helps limit individual variation. The mix of subjective and objective measures is a strength. However, the open-label approach may introduce bias. Diet and lifestyle are only monitored, which may confound results. The sample size may also be limited for detailed microbiome work. Despite these limitations, the suggested trial protocol offers a good framework for exploring gut-targeted interventions in this population.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The revised manuscript improved a lot, should be accepted for publication 

Author Response

We would like to thank you for taking the time to revise our manuscript and provide us with your valuable feedback which helped improve the quality of this work and to make it acceptable for publication.

Reviewer 2 Report

Comments and Suggestions for Authors

I thank the authors for their answers and clarifications. However, I do not agree with some of their points.

I understand that this is a randomized controlled crossover study. However, the prebiotic treatment will inevitably influence the gut microbiota and, consequently, the effects of the subsequent probiotic intervention. In fact, in some studies the prebiotics are administered before to restore gut dysbiosis. Therefore, the order of administration (prebiotic → probiotic or probiotic → prebiotic) is likely to affect gut microbiota composition and, in turn, gastrointestinal outcomes. While patients following a prebiotic diet will not be excluded, those receiving a probiotic diet will be excluded. Could the authors clarify how they plan to control for or rule out these potential carryover effects in order to clearly discern the effects attributable to each intervention?

Author Response

Thank you for this insightful comment. We agree that the sequence effect is a critical consideration in crossover trials. To mitigate potential bias from carryover effects, we have designed the study based on the following pillars of evidence from studies with healthy adults:

Ecological Resilience: We rely here on the principle of microbial resilience, where the unhealthy gut ecosystem resists permanent shifts from transient interventions (Fassarella et al., 2021). Data show that even with major dietary perturbations, the microbiome reverts to baseline within 2-3 days of cessation of the diet. (David et al., 2014)

Transience of Interventions and Washout: Systematic reviews indicate that probiotics typically do not colonize the gut and become mostly undetectable after stopping supplementation (Kristensen et al., 2016; Suez et al., 2018). Regarding the prebiotic component, the increase of Bifidobacterium and other beneficial taxa associated with oat bran and inulin-type fructans seemingly depends on continuous substrate intake; these levels return to baseline concentrations within 2-4 weeks post-supplementation (Connolly et al., 2016; Hughes et al., 2022).  Because the underlying neurogenic drivers of dysbiosis remain constant, the microbiome is expected to revert to its SCI-characteristic 'baseline' state once the exogenous prebiotic or probiotic pressure is removed (Bazzocchi et al., 2021). Our 4-week washout period represents a duration 1-2 times longer than the known biological persistence of these effects. The safety margin also considers the prolonged transit times of individuals with SCI compared to healthy adults and should therefore be absolutely sufficient to avoid potential carryover effects.

Furthermore, a statistical safeguard, to rigorously evaluate potential carryover effects, will be employed. Therefore, a four-tiered statistical approach will be applied:

1. Sequence Effect Analysis: We will include 'sequence' (AB vs. BA) as a fixed effect in our primary model. A significant sequence effect may indicate a carryover effect, baseline imbalance, or differential dropout in both sequences. A non-significant sequence effect indicates that the total response was independent of the order of administration.

2. Treatment-by-Period Interaction: We will test for an interaction between the intervention and the study period. A significant interaction would alert us that the probiotic's efficacy differed depending on whether it followed the prebiotic or not, i.e., it is an indication of a carryover or time-varying effect.

3. Pre-Period Baseline Comparison: We collect microbiome and clinical data at the start of both Period 1 and Period 2. We will statistically compare these two baselines to ensure the gut environment returned to its 'SCI-baseline' during the washout. Differences may indicate a carryover effect or a time trend

4. Fit model with carryover term: We will introduce a carryover term in the model that represents the effect of the treatment given in the previous period on the current period’s outcome. A significant carryover effect may indicate that washout was inadequate.

In the event that carryover effects cannot be ruled out, we will conduct a sensitivity analysis using only Period 1 data (treating the study as a parallel-group RCT). This approach ensures transparency and allows us to evaluate the robustness of our primary crossover findings. While we acknowledge this reduces statistical power, it provides an unbiased estimate of the intervention effect, free from any potential carryover effect. In lines 237-240 the wording of the statistical plan has been slightly adjusted to reflect this answer more accurately.

Also, a probiotic diet is not excluded per se if the probiotic is part of a normal diet (e.g. yoghurt) to reflect real life. People are only excluded if they specifically, additionally take a 'probiotic intervention' like the one we employ in the study, as this would not be part of a normal diet and would be classified as a 'medication' influencing the gut.

Bazzocchi, G., Turroni, S., Bulzamini, M. C., D’Amico, F., Bava, A., Castiglioni, M., Cagnetta, V., Losavio, E., Cazzaniga, M., Terenghi, L., De Palma, L., Frasca, G., Aiachini, B., Cremascoli, S., Massone, A., Oggerino, C., Onesta, M. P., Rapisarda, L., Pagliacci, M. C., Biscotto, S., Scarazzato, M., Giovannini, T., Balloni, M., Candela, M., Brigidi, P., & Kiekens, C. (2021). Changes in gut microbiota in the acute phase after spinal cord injury correlate with severity of the lesion. Scientific Reports, 11(1), 12743. https://doi.org/10.1038/s41598-021-92027-z

Connolly, M. L., Tzounis, X., Tuohy, K. M., & Lovegrove, J. A. (2016). Hypocholesterolemic and Prebiotic Effects of a Whole-Grain Oat-Based Granola Breakfast Cereal in a Cardio-Metabolic "At Risk" Population. Front Microbiol, 7, 1675. https://doi.org/10.3389/fmicb.2016.01675

David, L. A., Maurice, C. F., Carmody, R. N., Gootenberg, D. B., Button, J. E., Wolfe, B. E., Ling, A. V., Devlin, A. S., Varma, Y., Fischbach, M. A., Biddinger, S. B., Dutton, R. J., & Turnbaugh, P. J. (2014). Diet rapidly and reproducibly alters the human gut microbiome. Nature, 505(7484), 559-563. https://doi.org/10.1038/nature12820

Fassarella, M., Blaak, E. E., Penders, J., Nauta, A., Smidt, H., & Zoetendal, E. G. (2021). Gut microbiome stability and resilience: elucidating the response to perturbations in order to modulate gut health. Gut, 70(3), 595-605. https://doi.org/10.1136/gutjnl-2020-321747

Hughes, R. L., Alvarado, D. A., Swanson, K. S., & Holscher, H. D. (2022). The Prebiotic Potential of Inulin-Type Fructans: A Systematic Review. Adv Nutr, 13(2), 492-529. https://doi.org/10.1093/advances/nmab119

Kristensen, N. B., Bryrup, T., Allin, K. H., Nielsen, T., Hansen, T. H., & Pedersen, O. (2016). Alterations in fecal microbiota composition by probiotic supplementation in healthy adults: a systematic review of randomized controlled trials. Genome Med, 8(1), 52. https://doi.org/10.1186/s13073-016-0300-5

Suez, J., Zmora, N., Zilberman-Schapira, G., Mor, U., Dori-Bachash, M., Bashiardes, S., Zur, M., Regev-Lehavi, D., Ben-Zeev Brik, R., Federici, S., Horn, M., Cohen, Y., Moor, A. E., Zeevi, D., Korem, T., Kotler, E., Harmelin, A., Itzkovitz, S., Maharshak, N., Shibolet, O., Pevsner-Fischer, M., Shapiro, H., Sharon, I., Halpern, Z., Segal, E., & Elinav, E. (2018). Post-Antibiotic Gut Mucosal Microbiome Reconstitution Is Impaired by Probiotics and Improved by Autologous FMT. Cell, 174(6), 1406-1423.e1416. https://doi.org/10.1016/j.cell.2018.08.047

Round 3

Reviewer 2 Report

Comments and Suggestions for Authors

Dear authors,

Thank you for your reply. Please add a brief portion or summary of this information to the strenghts and limitations section of the study, in order to make readers aware of this phenomenom. I was also unable to find the edited version of the methods section regarding the statistical analysis mentioned in the rebuttal letter. Therefore, please ensure that this information is included in the revised manuscript.

I look forward to seeing the outcomes of this very intersting study.

Author Response

Thank you for your constructive feedback.

As requested, we have added a summary of the phenomenon to the Strengths and Limitations section (lines 298-305). Additionally, the Statistical Analysis section has been updated to explicitly state the approach for potential carryover effects (lines 237- 246).

We appreciate your interest in our study and believe these additions have strengthened the transparency of our protocol.