Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Summary: First, I wish to thank the Authors for addressing a clinically relevant and underexplored problem in feline medicine: the management of hospitalised feral cats. This prospective single-arm study evaluated repeated pregabalin administration in 25 cats, of which 20 completed the protocol, using pain, behavioural, sedation, physiological, rescue-analgesia, and salivary-cortisol outcomes. The repeated clinical assessments and inclusion of salivary cortisol are useful features. However, the absence of an untreated or placebo control prevents temporal improvements from being attributed specifically to pregabalin. In addition, the statistical analysis of repeated measurements is insufficiently described, several reported percentage changes are inconsistent with the tabulated means, and the reported ethics approval date appears to post-date study initiation.

Recommendation: Reject.

I declare no conflicts of interest in relation to this review.

General Critique

1. The study addresses an important welfare and clinical-management problem in a population that is difficult to investigate.
2. The main limitation is the uncontrolled design. Pregabalin exposure is completely confounded with hospital acclimatisation, repeated handling, concurrent treatment, disease evolution, and rescue analgesia. The study can therefore describe temporal changes during treatment, but it cannot establish a pregabalin-specific effect.

 Major Comments

• Lines 100-120 and 436-441: The study reportedly began in March 2024, whereas the Institutional Review Board Statement gives an approval date of 1 December 2024. Please clarify the definitive approval date, protocol number, approving committee, and whether any animals entered the research protocol before approval. Given the importance of this discrepancy, I recommend editorial verification.
• Lines 120-155 and Discussion: All cats received pregabalin, and baseline measurements were used as the comparator. Temporal changes therefore cannot distinguish a drug effect from acclimatisation, repeated operator exposure, treatment of the underlying disease, clinical procedures, or other time-dependent factors. Please substantially temper causal or efficacy statements throughout the Abstract, Discussion, and Conclusions and describe the study as exploratory and uncontrolled.
• Lines 207-222 and Tables 2-4: Several percentages cannot be reproduced from the tables. For example, FGS decreases from 3.25 to 1.40 at T36, corresponding to approximately 57%, not 18.5%. DSS shows similar discrepancies. Percentage change in GSS is also difficult to interpret because the scale crosses zero. Please recalculate all derived values from the original dataset and reconcile the Abstract, Results, tables, and figures.
• Lines 143-150: Anti-inflammatory and rescue analgesic drugs were permitted during the observation period. These interventions can directly influence pain, sedation, behaviour, and physiological outcomes. Please report the relevant drugs and timing for each cat and acknowledge this explicitly as a major source of confounding.

Statistics

• Report principal effects with 95% confidence intervals.
• The repeated observations within each cat are not independent. Please clarify whether the reported two-way ANOVA accounted for the within-cat repeated-measures structure and provide the complete model specification. If not, the analysis should be reconsidered using an appropriate repeated-measures or mixed-effects approach.

Minor Comments

Section	Original/Issue	Suggested correction
Abstract	Study dates differ from the Methods	Reconcile March 2024–September 2025 versus March 2024–April 2025.
Methods	“protocol number XXX”	Replace with the verified ethics approval number.
Throughout	American/British spelling is mixed	Use British English consistently: “behaviour”, “analysed”, etc.
Statistics	“P” and threshold reporting	Use italic lower-case p and exact values where possible.
Figures	“Graphic” and “Figure” used inconsistently	Use “Figure” consistently and check numbering.
Tables	Asterisks do not clearly identify comparisons	Define the comparator and statistical test in each footnote.
Cortisol groups	“healthy” cats	Avoid “healthy”. These were hospitalized cats without severe comorbidities.

Comments for author File: Comments.pdf

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript reports an uncontrolled prospective clinical study evaluating twice-daily oral pregabalin (10 mg/kg) over two days in 20 hospitalized feral cats, with behavioural, sedation and pain scoring at nine time points and salivary cortisol as an objective stress biomarker. The topic is clinically relevant and underexplored. The "off–on–off" temporal pattern of the scores is the strongest argument the authors possess, and the manuscript would benefit from exploiting it more rigorously. Please find bellow my comments

The Methods state that "the study protocol was approved by the Institutional Ethics Committee (protocol number XXX)" - the placeholder must be replaced with the actual committee name, approval number and date. The Institutional Review Board Statement describes an agreement between Orion Corporation and the University of Bari (protocol 2001-5 III/13, dated 1 December 2024); (is this a sponsorship agreement?) The study is stated to have begun in March 2024, whereas the only dated approval is 1 December 2024; the authors are asked to clarify the chronology. The study period is given as "March 2024 to September 2025" in the Abstract but "March 2024 and April 2025" in the Methods; the correct interval should be stated consistently.

The Informed Consent Statement ("informed consent was obtained from all subjects... written informed consent has been obtained from the patient(s)") is template text inapplicable to feral cats; it should be replaced with "Not applicable," or with a statement regarding colony caretakers if consent was in fact obtained from them.

The study was funded by Orion (grant 2001-5 III/13), which is also the manufacturer of the product under investigation (Bonqat®), yet the Conflicts of Interest section states that "the authors declare no conflicts of interest." The authors are asked to revise the statement accordingly and to specify the sponsor's role (or absence thereof) in study design, data analysis and the decision to publish.

All cats were admitted acutely ill, evaluated at peak novelty stress (T-Pre within 24 h of admission), and simultaneously received treatment for their underlying conditions — the Methods explicitly permit concurrent medication "including anti-inflammatory drugs." Consequently, improvements in FGS, DSS and GSS over four days may reflect environmental acclimatization and resolution of the primary disease as much as pregabalin. The Discussion's statement that "the findings confirm that the inhibitory mechanism of pregabalin provides behavioural and analgesic benefits" (lines 316–319) overstates what an uncontrolled design can show; "confirm" should be replaced with "suggest" or "are consistent with." The authors' strongest counter-argument — the partial rebound of all three scores during washout, resembling an off–on–off response — deserves formal support: it is suggested that the authors statistically compare T36 versus T48/T60 and report these contrasts explicitly, since a significant worsening upon drug withdrawal, at a time when acclimatization and disease treatment would predict continued improvement, is the one pattern the confounders cannot easily explain. In addition, the limitations passage should state which concomitant anti-inflammatory treatments were actually administered and in how many cats.

The Results state that HR "remained generally stable," yet Table 1 shows a decrease from 164.8 ± 21 bpm at T-Pre to 100 ± 25 bpm at T4 — a reduction of approximately 40%, which is neither stable nor clinically trivial — and the Discussion asserts that pregabalin acted "without significantly affecting physiological parameters such as HR" (line 318), contradicting the Abstract's own statement that HR decreased from T4 to T48. Table 1 carries no significance markers. The authors are asked to report the statistical results for HR and RR, reconcile the three statements, and discuss the HR reduction on its merits — it is possible that it reflects attenuation of sympathetic stress activation, which would in fact support, rather than weaken, the anxiolytic hypothesis, and comparison with the echocardiographic data of Li et al. (23) would be pertinent here.

Eight of twenty cats (40%) received rescue analgesia at T-Pre, i.e., opioid exposure preceded the first post-treatment evaluations; moreover, tramadol and buprenorphine appear in the evaluation-shifting rule although the stated rescue protocol specifies methadone only, and the Methods simultaneously state that "the administration of any medications... was permitted" and that "analgesic and sedative drugs were not allowed... in the period of study." The authors should: reconcile these statements;  report which rescue drugs and doses were actually administered, at which time points, and to how many cats; acknowledge in the limitations that opioid carryover may have influenced early scores despite the shifted evaluations; and consider presenting Table 5 alongside the FGS data, since the declining rescue requirement is itself a clinically meaningful outcome and currently underexploited.

 

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Dear Authors,

the study addresses a clinically relevant problem about the management of hospitalized feral cats and the potential role of pregabalin in reducing pain, fear, behavioural difficulties and the need for additional pharmacological intervention. The prospective assessment of behavioural scores together with salivary cortisol is interesting. However, some issues need to be addressed to improve the manuscript.

Abstract

Lines 33-35 and lines 103-104: The study period is reported as March 2024-September 2025 in the Abstract, whereas the Methods report March 2024-April 2025.

Lines 41-50: The statement that FGS, DSS and GSS “significantly decreased” is problematic, particularly for GSS. According to the scale description later in the manuscript, positive GSS values indicate increasing sedation, whereas negative values indicate increasing alertness. The numerical results therefore do not support the statement that GSS decreased as an indicator of improvement.

Lines 43-45: The reported percentage changes should be carefully checked against the corresponding baseline and follow-up values. For example, the FGS decreases from 3.25 at baseline to 1.40 at T36, corresponding to approximately a 56.9% reduction rather than 18.5%. Similar discrepancies appear to be present for the DSS values. Therefore, recalculate all percentage changes consistently throughout the manuscript, including the corresponding tables and graphics.

Introduction

Lines 87-99: The rationale for assessing pain, behaviour, sedation and stress would benefit from a more descriptive explanation based on the current literature. In particular, clarify the specific purpose of each assessment scale/biomarker in the context of hospitalized feral cats and explain why these parameters were considered relevant to the objectives of the study.

Materials and Methods

Lines 102-104: There is a clear inconsistency in the ethical approval information. The protocol number is reported as “XXX” in line 103, whereas the later Institutional Review Board statement identifies protocol 2001-5 III/13 dated 1 December 2024 (lines 436-440). This needs to be corrected.

Lines 112-114: The use of the DSS to support the classification of cats as feral is relevant, so I suggest clarifying how the threshold of DSS ≥9/25 was selected according to the cited reference, so that the basis for this inclusion criterion is clear to the reader.

Lines 142-146: Since concomitant medical treatments were permitted according to the clinical condition of each cat, the authors can briefly clarify whether any concomitant treatments could have influenced pain or behavioural assessments and should acknowledge this as a potential confounding factor.

Lines 146-150: Rescue analgesia was administered according to the FGS score, and subsequent assessments were scheduled according to the analgesic administered. I suggest a brief explanation of how FGS assessments performed after rescue analgesia were considered in the analysis, as analgesic administration itself could influence subsequent pain scores.

Lines 151-155: Because propofol was administered when additional sedation was required, clarify how cats receiving propofol were considered in the behavioural and sedation assessments. In particular, the statement that propofol had “minimal interference with the study assessments” can benefit from brief clarification.

Lines 158-160: The interpretation of the GSS requires substantial clarification. The authors state that scores from 0 to +3 indicate increasing sedation, whereas scores from 0 to -3 indicate increasing alertness. The results subsequently describe a “decrease” in GSS despite the observed shift from -1.05 at baseline to positive values during treatment. This appears inconsistent with the scale itself, so check carefully the GSS results and discussion.

Lines 166-170: The DSS is described as a behavioural scale with five categories, but the directionality of the total score is not sufficiently explained. Explicitly state whether higher scores indicate greater fear/aggression and whether lower scores represent improved behavioural responses. This is essential for interpreting the reported reductions.

Results

Lines 210-220: The presentation of the statistical results for FGS, DSS and GSS could be made clearer. For the comparisons described as significant in the text, provide the corresponding P values or clearly specify which time-point comparisons reached statistical significance, rather than relying exclusively on asterisks in the tables and figures.

Lines 229-231: The classification of cats into “without severe comorbidities” and “with severe comorbidities” could be described more clearly in the Materials and Methods. I suggest explaining the clinical criteria used to assign cats to these groups, indicating how this classification was established.

Figures, Tables and Schemes

Figures 1-2: The individual data-sheet figures provide mainly a description of the study recording form. The authors may wish to consider whether these figures are necessary in the main manuscript and whether a concise study timeline or schematic representation of the assessment schedule would provide more useful information for the reader.

Tables 2-4: Carefully check all numerical calculations and percentage changes reported in these tables and in the corresponding graphics.

Discussion

Lines 319-330: The interpretation of the DSS and FGS should be more cautious. In particular, the statement that the FGS demonstrates a reduction in acute pain could recognise that pain scores may also change because of clinical recovery, concomitant treatment and repeated handling.

Lines 331-332: The explanation that the authors simply considered pain at each time point to be “acute pain” is not entirely convincing. The cats had heterogeneous underlying disorders, some potentially associated with persistent or chronic pain. The authors could recognise that FGS measures acute pain expression at the time of assessment and does not establish the underlying temporal nature of the pain condition.

Lines 333-341: The interpretation of GSS needs correction. The manuscript itself acknowledges that positive values represent increasing sedation. Therefore, the movement from a baseline of -1.05 to positive values during treatment should not be described as a reduction in sedation. More importantly, the authors do not distinguish pharmacological sedation from reduced fear or improved tolerance of the operators using this score alone.

Lines 342-351: The interpretation of the lowest scores at T36 as evidence of a cumulative pregabalin effect is not sufficiently supported. The same temporal pattern could result from habituation to hospitalization, clinical improvement, repeated feeding/handling by familiar operators, or changes in concomitant treatment. I suggest describing better this interpretation.

Lines 360-374: The interpretation of the cortisol findings in terms of HPA-axis activity should be slightly more cautious, since only salivary cortisol was measured. The observed differences between cats with different clinical severity are interesting, but the manuscript could acknowledge that changes in cortisol may also reflect differences in disease severity and clinical evolution.

Lines 397-407: The limitations section can be expanded to acknowledge the main factors that may affect interpretation of the observed temporal changes, particularly the absence of a comparator group, the heterogeneous clinical conditions, concomitant treatments, and the limited sample size.

Funding, Ethics and Conflict of Interest

Lines 436: Since the study was supported by Orion and the investigated product was Bonqat (Orion Corporation), explain whether the sponsor had any involvement in the study design, data analysis, interpretation of the results, manuscript preparation, or the decision to submit the manuscript.

Lines 442-443: The informed consent statement appears to use terminology more commonly applied to human studies. Since the study involved feral cats, I suggest explaining who provided authorization for the animals to undergo the procedures and treatment, and revise the statement using terminology appropriate for veterinary research.

Line 448: Given the study funding by Orion and the use of an Orion product, the Conflict of Interest statement can be clarified to ensure consistency with the Funding statement.

Kind regards

 

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

I would like to sincerely thank the Authors for their considerable efforts and the excellent work undertaken in revising the manuscript.

All my previous concerns have been satisfactorily addressed, and I have no further comments or requests for revision. I would also like to extend my congratulations and best wishes to those Authors currently pursuing their PhDs, and wish them continued success in their doctoral research and future academic careers.

Comments for author File: Comments.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Dear Authors,

Thank you for the careful revision of the manuscript and for addressing all the comments raised in the previous review. The revised version has been improved, and the issues highlighted in the first round have been adequately addressed.

Kind regards