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Effect and mechanism of inhibition of PI3K/Akt/mTOR signal pathway on chronic neuropathic pain and spinal microglia in a rat model of chronic constriction injury · 10.18632/oncotarget.17629

Name: Verified Rigor Record — Effect and mechanism of inhibition of PI3K/Akt/mTOR signal pathway on chronic neuropathic pain and spinal microglia in a rat model of chronic constriction injury · 10.18632/oncotarget.17629
Item: Effect and mechanism of inhibition of PI3K/Akt/mTOR signal pathway on chronic neuropathic pain and spinal microglia in a rat model of chronic constriction injury · 10.18632/oncotarget.17629
Rating: 75
Author: Αlpha¹

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Αlpha¹ Editorial

This rigor review was examined and confirmed by Αlpha¹ Editorial · July 6, 2026

Αlpha¹ Rigor Review

Rigor Reviewed

WARN

Warnings to address · Interventional Study

Eight dimensions — click a segment for details

Critical

High-priority

Model-reported confidence: highReviewer agreement: moderateEngine: 7.6.0Source: full text

Not verified in this report

No reported statistical tests were found to recompute.
No data or code availability links were detected to verify.

This Αlpha¹ Rigor Review uses AI Rigor Reviewers trained on a curated corpus of high-fidelity and retracted papers, with expert supervision and curation. It can still make mistakes; verify each finding against the source before relying on it.

This preclinical rat study on PI3K inhibition in neuropathic pain has a sound scientific premise and adequate ethics approval, but suffers from critical gaps in reporting that undermine confidence in its conclusions. Study design details (randomization method, blinding, power analysis) are missing, key resources lack catalog/RRID identifiers, statistical reporting is minimal (no exact p-values or effect sizes), and no data are shared. Copyedit issues include a major terminology inconsistency ('marrow' vs. 'spinal cord') and several typos.

All eight dimensions were evaluated based on the full-text manuscript. The statistics verification component found no tests to recompute (coverage note: only threshold p-values reported). The citation check found 0 retracted or not-found references. The study is preclinical interventional (animal model), so human-subject dimensions are not applicable. Three independent reviewers' evaluations were synthesized; divergence occurred on study design (2 warn, 1 fail) and reporting transparency (2 warn, 1 pass) and was resolved by applying the scoring rules to the combined checklist evidence.

12 major claims checked against the paper's own evidence: all adequately supported.

3 integrity concerns flagged (0 high).

Rigor

Eight Dimensions3 pass · 4 warn · 1 fail

12 major claims3 partial9 supported

The introduction cites prior research on PI3K/Akt/mTOR in CNP and cancer pain, acknowledges limitations in current knowledge, and formulates a clear hypothesis. Prior work is cited with both strengths and weaknesses addressed (e.g., 'few have been reported about the impact of the signal pathway on activation of spinal microglia'). The premise_rationale is strong: 'we hypothesize that PI3K/Akt/mTOR pathway may be involved in the progression of CNP.' Limitations of prior research are explicitly discussed: 'previous studies have disclosed the relationship between PI3K/Akt pathway and CNP, however, few have been reported about the impact of the signal pathway on activation of spinal microglia.' The study design directly addresses this gap.

Sub-criteria

prior work citedADEQUATE

premise rationaleADEQUATE

limitations addressedADEQUATE

Per reviewer (3) — the votes behind the verdict

PASSReviewer 1· 90% conf

The paper establishes a clear scientific premise by citing prior work on PI3K/Akt/mTOR pathway in pain and microglia, and provides a logical rationale for the study.

Evidence

direct quote[Introduction, paragraph 4]

“a rat model of bone cancer pain demonstrated that PI3K inhibitor reduced Akt expression and alleviated the mechanical allodynia in the rats through stimulating spinal microglia”

direct quote[Introduction, final paragraph]

“few have been reported about the impact of the signal pathway on activation of spinal microglia, an important factor in pathogenesis of CNP”

absence[Introduction]

Limitations of prior work are not explicitly addressed in the study design.

PASSReviewer 2· 90% conf

The scientific premise is well-established with adequate prior work cited, a logical rationale, and acknowledged limitations.

Evidence

direct quote[Introduction, paragraphs 1-3]

“Recent studies reported that by cell cycle inhibition, hyperesthesia after spinal cord injury could be reduced... PI3K/Akt/mTOR pathway closely associates with transition of G1-S phase of cell cycle.”

direct quote[Introduction, last paragraph]

“Therefore, in the present study, we built a rat model of chronic constriction injury (CCI) to investigate the effect and mechanism of PI3K/Akt/mTOR signal pathway on the progression of CNP and on activation of spinal microglia as well.”

direct quote[Discussion, last paragraph]

“However, the present study is preliminary. The exact mechanism of PI3K/Akt/mTOR signal pathway modulating microglia activation remains to be discussed in the future studies.”

PASSReviewer 3· 95% conf

The paper adequately cites prior work establishing the premise, provides a logical rationale linking PI3K/Akt/mTOR signaling to chronic neuropathic pain and microglial activation, and identifies gaps in prior research.

Evidence

direct quote[Introduction, paragraph 4]

“Previous studies have disclosed the relationship between PI3K/Akt pathway and CNP, however, few have been reported about the impact of the signal pathway on activation of spinal microglia, an important factor in pathogenesis of CNP.”

direct quote[Introduction, paragraph 4]

“we hypothesize that PI3K/Akt/mTOR pathway may be involved in the progression of CNP.”

direct quote[Abstract & Introduction, paragraph 4]

The study is a controlled intervention with six groups including sham, CCI, vehicle (DMSO), and a positive control. The randomization method is vaguely described: 'These rats were randomly assigned into 6 groups' without stating the method (e.g., random number generator, stratification). Blinding is not mentioned at all; for a preclinical study with behavioral and histology endpoints, this is a significant gap. No a priori power analysis or sample size calculation is reported. The distinction between biological and technical replicates is not discussed. Inclusion/exclusion criteria are not pre-specified (only a statement about post-operative infection/mutilation is noted). Outlier handling is not addressed. Controls are adequate (sham, vehicle, positive control). No independent replication is reported or mentioned. The CCI model itself follows an established method.

Sub-criteria

controlsADEQUATE

power analysisNOT REPORTED

blinding levelsNOT REPORTED

outlier handlingNOT REPORTED

randomization unitADEQUATE

inclusion exclusionINADEQUATE

randomization methodINADEQUATE

replicate distinctionNOT REPORTED

independent replicationNOT REPORTED

Per reviewer (3) — the votes behind the verdict

WARNReviewer 1· 80% conf

The study design is generally appropriate, but key rigor features such as randomization method, blinding, and power analysis are not reported, and sample sizes are small.

Evidence

direct quote[Materials and Methods, Animal treatment and grouping]

“These rats were randomly assigned into 6 groups (n =18 in each group)”

absence[Materials and Methods]

Blinding is not reported anywhere in the paper.

absence[Materials and Methods]

No power analysis or sample size justification is provided.

FAILReviewer 2· 85% conf

Study design lacks critical rigor elements: no randomization method, no blinding, no power analysis, no inclusion/exclusion criteria, no outlier handling, and no independent replication.

Evidence

direct quote[Materials and Methods, Animal treatment and grouping]

“These rats were randomly assigned into 6 groups”

absence[Entire manuscript]

Blinding is not mentioned.

absence[Entire manuscript]

A priori power analysis is not reported.

WARNReviewer 3· 85% conf

The study design is generally adequate as a preclinical interventional study, but several important rigor elements are missing or inadequately reported, notably the lack of blinding, power analysis, clear replicate distinction, pre-specified inclusion/exclusion criteria, and outlier handling.

Evidence

direct quote[Materials and Methods, Animal treatment and grouping]

“These rats were randomly assigned into 6 groups”

absence[All sections]

Blinding is not reported at any stage.

absence[All sections]

No a priori power or sample size calculation is reported.

Sex, age, weight, health status, species, strain, source, and housing conditions are all reported. Sex justification for using only males is not provided, but the study is a proof-of-concept mechanism study.

Sub-criteria

demographicsN/A

sex reportedADEQUATE

sex justifiedNOT REPORTED

age weight healthADEQUATE

housing conditionsADEQUATE

species strain sourceADEQUATE

Per reviewer (3) — the votes behind the verdict

PASSReviewer 1· 85% conf

Biological variables are adequately reported for a rat study: sex, age, weight, and source are given, and housing conditions are described.

Evidence

direct quote[Materials and Methods, Animal treatment and grouping]

“Male Sprague Dawley (SD) rats (n = 108, specific pathogen free (SPF)) were gained from the Laboratory Animal Center of Shandong University (age range: 16∼20 weeks; weight range: 220 ∼ 260 g).”

direct quote[Materials and Methods, Animal treatment and grouping]

“The rats were caged (5 rats/cage) at a temperature of 20 ∼ 26°C a humidity of 40 ∼ 70% and an illumination cycle of 12 hours/day and they were free to take water and food.”

absence[Materials and Methods, Animal treatment and grouping]

No scientific justification is given for using only male rats.

PASSReviewer 2· 90% conf

Biological variables are adequately reported for sex, age/weight, species/strain/source, and housing conditions; sex justification is missing but not critical given the study design.

Evidence

direct quote[Materials and Methods, Animal treatment and grouping]

“Male Sprague Dawley (SD) rats”

direct quote[Materials and Methods, Animal treatment and grouping]

“age range: 16∼20 weeks; weight range: 220 ∼ 260 g; specific pathogen free (SPF)”

direct quote[Materials and Methods, Animal treatment and grouping]

“caged (5 rats/cage) at a temperature of 20 ∼ 26°C a humidity of 40 ∼ 70% and an illumination cycle of 12 hours/day”

WARNReviewer 3· 90% conf

Sex of animals is reported (male only) but not justified; age, weight, species, strain, source, and housing conditions are adequately described. Demographics are not applicable (animal study).

Evidence

direct quote[Materials and Methods, Animal treatment and grouping]

“Male Sprague Dawley (SD) rats”

direct quote[Materials and Methods, Animal treatment and grouping]

The study states approval by the Institutional Animal Care and Use Committee of Gongli Hospital, Second Military Medical University, and compliance with the NIH Guide for the Care and Use of Laboratory Animals. Both applicable sub-criteria are adequate.

Sub-criteria

iacuc statementADEQUATE

informed consentN/A

irb ethics statementN/A

regulatory complianceADEQUATE

Per reviewer (3) — the votes behind the verdict

PASSReviewer 1· 90% conf

Ethical approval is adequately reported: a named IACUC approved the protocol, and compliance with a recognized guideline is stated.

Evidence

direct quote[Materials and Methods, Ethic statement]

“The protocol was approved by the Institutional Animal Care and Use Committee of Gongli Hospital, Second Military Medical University.”

direct quote[Materials and Methods, Ethic statement]

“The present study was conducted in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.”

PASSReviewer 2· 95% conf

Ethical approvals are adequately reported with named animal welfare committee approval and regulatory compliance.

Evidence

direct quote[Materials and Methods, Ethic statement]

“The protocol was approved by the Institutional Animal Care and Use Committee of Gongli Hospital, Second Military Medical University.”

direct quote[Materials and Methods, Ethic statement]

“The present study was conducted in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.”

PASSReviewer 3· 95% conf

The study reports an IACUC approval from a named institution and states compliance with a recognized guideline, meeting the criteria for an animal study.

Evidence

direct quote[Materials and Methods, Ethic statement]

“The protocol was approved by the Institutional Animal Care and Use Committee of Gongli Hospital, Second Military Medical University.”

direct quote[Materials and Methods, Ethic statement]

“The present study was conducted in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.”

Antibodies are reported with vendor and dilution, but not RRIDs. The drug wortmannin is identified by vendor but no catalog number or lot number is given for the drug itself. Primer sequences are provided. Software such as 'SPSS 21.0', 'ImageJ', and 'ImageProPlus' are named, but versions for the latter two are missing. No custom code was used, so code sharing is n/a. Cell lines and mycoplasma testing are not applicable to this animal tissue study.

Sub-criteria

mycoplasma testingN/A

reagents identifiedINADEQUATE

organisms identifiedADEQUATE

antibodies identifiedINADEQUATE

cell line authenticationN/A

software tools identifiedINADEQUATE

Per reviewer (3) — the votes behind the verdict

WARNReviewer 1· 85% conf

Antibodies and some reagents are identified with vendors and catalog numbers, but code, cell lines, and mycoplasma testing are not applicable, and software is only partially identified.

Evidence

direct quote[Materials and Methods, Western blotting]

“The primary rabbit-antiPI3K antibody (diluted at 1:1000) and rabbit-anti-p-Akt antibody (diluted at 1:1000) were bought from the Abcam (UK)”

direct quote[Materials and Methods, Animal treatment and grouping]

“wortmannin (Shanghai Solarbio Science & Technology Co., Ltd) (0.5 mg/kg)”

direct quote[Materials and Methods, Immunohistochemistry and Immunofluorescence]

“ImageJ, image-analyzing software, was used to analyze optical densities”

FAILReviewer 2· 80% conf

Key resources are inadequately identified; most antibodies and reagents lack catalog numbers or RRIDs, and software tools lack full versioning.

Evidence

direct quote[Materials and Methods, Western blotting]

“The primary rabbit-antiPI3K antibody (diluted at 1:1000) and rabbit-anti-p-Akt antibody (diluted at 1:1000) were bought from the Abcam (UK); and the primary rabbit-anti-mTOR antibody (diluted at 1:1000), rabbit-anti-P70S6K antibody (diluted at 1:1000) and rabbit-anti-GAPDH antibody (diluted at 1:1000) were from the Santa Cruz (USA).”

direct quote[Materials and Methods, Animal treatment and grouping]

“wortmannin (Shanghai Solarbio Science & Technology Co., Ltd) (0.5 mg/kg)”

direct quote[Materials and Methods, Animal treatment and grouping]

“Male Sprague Dawley (SD) rats (n = 108, specific pathogen free (SPF)) were gained from the Laboratory Animal Center of Shandong University”

WARNReviewer 3· 85% conf

Key resources are identified but with important omissions: antibodies lack catalog numbers/RRIDs, the key drug (wortmannin) is identified with a vendor but without formulation details, and primer sequences are provided but software versions are missing.

Evidence

direct quote[Materials and Methods, Western blotting]

“The primary rabbit-antiPI3K antibody (diluted at 1:1000) and rabbit-anti-p-Akt antibody (diluted at 1:1000) were bought from the Abcam (UK)”

direct quote[Materials and Methods, Table 3]

“Table 3 Primers sequences of PI3K, Akt, mTOR and GAPDH used in quantitative real-time polymerase chain reaction (qRT-PCR)”

direct quote[Materials and Methods, Animal treatment and grouping]

“wortmannin (Shanghai Solarbio Science & Technology Co., Ltd) (0.5 mg/kg)”

The paper names t-tests and one-way ANOVA as the statistical methods. However, no test assumptions (normality, equal variance) are reported or verified. Exact p-values are not given; only inequality statements like 'P < 0.05' are used. No effect sizes or confidence intervals are reported. SPSS 21.0 is identified as the software. Data presentation includes error bars (SD) and per-group n, but individual data points are not shown. Mathematical plausibility cannot be fully verified without exact p-values or raw data, but no obvious arithmetic errors are detected in the reported means and SDs.

Sub-criteria

tests namedADEQUATE

exact p valuesNOT REPORTED

effect sizes ciNOT REPORTED

data presentationINADEQUATE

software identifiedADEQUATE

assumptions verifiedNOT REPORTED

mathematical plausibilityN/A

Per reviewer (3) — the votes behind the verdict

WARNReviewer 1· 85% conf

Statistical methods are named but incompletely reported: assumptions are not verified, effect sizes are missing, and exact p-values are not reported (only thresholds).

Evidence

direct quote[Materials and Methods, Statistical analysis]

“Comparisons within group were performed by t test while between groups by one-way analysis of variance (ANOVA).”

direct quote[Results, e.g., Table 1 footnote]

“*, P < 0.05 in comparison with the control group”

absence[Results and Statistical analysis]

No effect sizes or confidence intervals are reported.

WARNReviewer 2· 85% conf

Statistical tests and software are named, but assumptions are not verified, exact p-values are not reported, effect sizes/confidence intervals are absent, and data presentation lacks individual data points.

Evidence

direct quote[Materials and Methods, Statistical analysis]

“Comparisons within group were performed by t test while between groups by one-way analysis of variance (ANOVA).”

direct quote[Throughout Results]

“all P < 0.05”

absence[Figures]

Individual data points are not shown.

WARNReviewer 3· 80% conf

Statistical tests are named, software is identified, and per-group n and error bars are reported, but test assumptions are not verified, exact p-values are not reported (only thresholds), effect sizes/confidence intervals are absent, and individual data points are not shown.

Evidence

direct quote[Materials and Methods, Statistical analysis]

“Comparisons within group were performed by t test while between groups by one-way analysis of variance (ANOVA). P < 0.05 indicated statistical significance.”

direct quote[Results, Tables 1 & 2 and throughout]

“all P < 0.05”

direct quote[Materials and Methods, Statistical analysis]

“SPSS 21.0 software was used for data analysis.”

The paper does not include any data availability statement, nor are any raw data deposited in a public repository. No custom code was developed, so code sharing is not applicable. For a preclinical study, raw data (e.g., Western blot images, qRT-PCR data) should be made available; their absence is a significant gap.

Sub-criteria

code sharingN/A

accession numbersN/A

repository depositNOT REPORTED

data availability statementNOT REPORTED

Per reviewer (3) — the votes behind the verdict

FAILReviewer 1· 95% conf

No data availability statement, repository deposit, or code sharing is provided; the paper completely lacks open science practices.

Evidence

absence[Entire paper]

No data availability statement is present anywhere in the paper.

absence[Entire paper]

No repository deposit or accession numbers are reported.

FAILReviewer 2· 80% conf

No data availability statement is provided, raw data are not deposited, and code is not shared.

Evidence

absence[Entire manuscript]

No data availability statement present.

absence[Entire manuscript]

No repository deposit mentioned.

FAILReviewer 3· 95% conf

No data availability statement, no repository deposit, no accession numbers, and no code sharing is reported. This is a significant gap for a contemporary study.

Evidence

absence[Entire manuscript]

No data availability statement is present.

absence[Entire manuscript]

No repository deposit or accession numbers reported.

absence[Entire manuscript]

No code sharing is reported.

Methods are fairly complete: animal handling, CCI model, drug administration, behavioral tests, molecular assays, and statistical analysis are described. Trial registration is not applicable (animal study). No reporting guideline (e.g., ARRIVE) is mentioned. All outcomes: the paper reports positive and negative (non-significant) results for some comparisons (e.g., 'no statistical difference among the CCI, sham and control groups was identified in their food intake, water intake and body weight'), but negative results are not systematically reported for all measures. Limitations are discussed in one sentence: 'the present study is preliminary. The exact mechanism of PI3K/Akt/mTOR signal pathway modulating microglia activation remains to be discussed in the future studies.' Conclusions are proportional: 'Inhibition of PI3K/Akt/mTOR signal pathway may alleviate CNP and reduce microglia and GFAP and NGF expressions in marrow in a rat model of CCI.' The 'may' appropriately qualifies the claim. Funding and conflicts: Funding is reported: 'This work was funded by Key Discipline Group Construction Project of Pudong Health Bureau of Shanghai (PWZxq2014-06).' Conflicts: 'The authors have declared that no competing interests exist.'

Sub-criteria

funding coiADEQUATE

trial registrationN/A

reporting guidelineNOT REPORTED

methods completenessADEQUATE

all outcomes reportedINADEQUATE

limitations discussedINADEQUATE

conclusions proportionalADEQUATE

Per reviewer (3) — the votes behind the verdict

WARNReviewer 1· 80% conf

Methods are fairly complete for replication, but no reporting guideline is followed, negative results are not fully reported, and the conclusion somewhat overstates the evidence.

Evidence

absence[Materials and Methods]

No reporting guideline (e.g., ARRIVE) is referenced.

direct quote[Discussion, final paragraph]

“the present study is preliminary. The exact mechanism of PI3K/Akt/mTOR signal pathway modulating microglia activation remains to be discussed in the future studies.”

direct quote[Footnotes]

“The authors have declared that no competing interests exist. This work was funded by Key Discipline Group Construction Project of Pudong Health Bureau of Shanghai (PWZxq2014-06).”

PASSReviewer 2· 90% conf

Reporting is largely transparent with detailed methods, all outcomes reported, proportional conclusions, and funding/COI disclosure; only a reporting guideline checklist is missing.

Evidence

paraphrase[Materials and Methods]

“The methods section includes detailed protocols for CCI model, drug administration, behavioral tests, qRT-PCR, Western blot, IHC, and immunofluorescence.”

absence[Entire manuscript]

Reporting guideline not referenced.

direct quote[Funding and COMPETING INTERESTS sections]

“This work was funded by Key Discipline Group Construction Project of Pudong Health Bureau of Shanghai (PWZxq2014-06).”

WARNReviewer 3· 85% conf

Methods are detailed enough for replication, funding and conflicts are reported, limitations are briefly discussed, and conclusions are generally proportional. However, no reporting guideline is referenced, all outcomes are not clearly pre-specified with negative results, and the trial is not registered (not applicable for animal study).

Evidence

paraphrase[Materials and Methods, all subsections]

“The methods section includes detailed descriptions of animal treatment, CCI model, drug administration, behavioral tests, qRT-PCR, western blotting, immunohistochemistry, and immunofluorescence.”

absence[Entire manuscript]

No reporting guideline (e.g., ARRIVE) is mentioned.

direct quote[Discussion, last paragraph]

“However, the present study is preliminary. The exact mechanism of PI3K/Akt/mTOR signal pathway modulating microglia activation remains to be discussed in the future studies.”

22 copyedit issues flagged (3 major): mostly typo, consistency, clarity.

Checked 36 references: 35 verified — 1 not checked.

Αlpha¹ Rigor Action ItemsFinal pass before journal submission

This is a post-publication audit. The published work has a reasonable premise but the reporting gaps — particularly the absence of blinding, power analysis, exact p-values, and data availability — are substantial enough that an informed reader should weigh them carefully. The consistent use of 'marrow' for spinal cord tissue (a likely mistranslation) and the OX-4/OX-42 typo in the abstract are confusing. An erratum or correction would be warranted to clarify the tissue terminology and the antibody target. Independent re-analysis would be needed to verify the reported statistical claims, as no raw data are available.

1.
HIGHdata code
Add a Data Availability Statement specifying whether raw data (Western blot images, qRT-PCR data, behavioral scores) are available and from whom or where (e.g., a repository).
This is a data-driven preclinical study; the complete absence of any data sharing statement is a major transparency gap that limits reproducibility.
2.
HIGHreporting
Correct the terminology in the Results: replace all instances of 'marrow' with 'spinal cord' or 'spinal tissue' to match the Methods (L4-L6 spinal cord collection).
The repeated use of 'marrow' (typically meaning bone marrow) for spinal cord tissue is seriously confusing and undermines the reader's ability to interpret the findings; this should be addressed in a correction.
3.
HIGHcopyedit
Fix the typo in the Abstract: change 'OX-4' to 'OX-42' to be consistent with the Methods and Results.
OX-4 is not used anywhere else in the paper; this appears to be a simple typo but creates an inconsistency that could confuse readers.
4.
HIGHrigor
Report the specific randomization method used (e.g., random number table) in the 'Animal treatment and grouping' section.
Simply stating 'randomly assigned' is insufficient; the method must be described to allow assessment of selection bias risk.
5.
HIGHrigor
Describe blinding procedures for surgery and outcome assessment in the Methods (who was blinded, at what stage).
Blinding is a critical rigor element for preclinical studies; its absence is a major limitation that should be disclosed and justified.
6.
HIGHrigor
Add an a priori power analysis or sample size justification for the primary behavioral outcomes, specifying the expected effect size and desired power.
Without a power analysis, the sample size (n=18/group) cannot be evaluated for adequacy, and the study may be underpowered for some comparisons.
7.
HIGHstatistics
Report exact p-values (e.g., 'P = 0.023') instead of only 'P < 0.05' throughout the Results section and Tables.
Threshold-only p-values prevent readers from assessing the strength of evidence and are not recomputable for verification.
8.
HIGHstatistics
Include effect sizes (e.g., Cohen's d) and 95% confidence intervals for key pairwise comparisons in the Results.
Effect sizes and CIs provide information about the magnitude and precision of effects, which p-values alone do not convey.
9.
HIGHreporting
List all pre-specified outcome measures and indicators (behavioral, molecular) and systematically state results (significant or not) for each in the Results.
The paper does not clearly define all outcomes upfront, and negative results are not systematically reported, risking selective reporting bias.
10.
MEDIUMrigor
Provide a justification for using only male rats (e.g., to avoid estrous cycle variability, or reference to prior work) in the Methods or Discussion.
Single-sex animal studies should be justified to inform generalizability; this is a standard expectation in contemporary pain research.
11.
MEDIUMother
Add catalog numbers for all antibodies from Abcam and Santa Cruz, and add RRIDs where available, in the Western blotting section.
Catalog numbers and RRIDs ensure unambiguous identification of key reagents and facilitate reproducibility.
12.
MEDIUMother
Add the catalog number or lot number for wortmannin (Shanghai Solarbio) in the 'Animal treatment and grouping' section.
Without a catalog number, the specific formulation and batch cannot be identified, affecting reproducibility.
13.
MEDIUMother
Provide version numbers for ImageJ and ImageProPlus in the immunohistochemistry and western blotting Methods.
Software versions are necessary for reproducibility of image analysis workflows.
14.
MEDIUMreporting
Reference the ARRIVE guidelines for preclinical animal research and include the completed checklist as supplementary material.
Following a reporting guideline ensures comprehensive and transparent reporting; many journals now require this.
15.
MEDIUMstatistics
Specify which post-hoc test was used for pairwise comparisons following one-way ANOVA in the Statistical analysis section.
The paper reports multiple pairwise comparisons but does not name the post-hoc test, making it impossible to assess correction for multiple comparisons.
16.
MEDIUMcopyedit
Fix the typo '4% paraformal dehyde' to '4% paraformaldehyde' in the Immunohistochemistry Methods.
This is a clear misspelling that should be corrected for professional presentation.
17.
MEDIUMcopyedit
Fix the typo 'fist' to 'first' in the Western blotting Methods (voltage description).
Typographical error that should be corrected.
18.
LOWcopyedit
Fix the duplicated word 'from from' in the Immunohistochemistry Methods (3% H2O2 source).
Minor typo.
19.
LOWcopyedit
Standardize decimal places in Table 2 (change '15.4 ± 3.80' to '15.40 ± 3.80' for consistency with other entries).
Inconsistent decimal places within a table detract from professional presentation.
20.
LOWcopyedit
Correct the spelling 'bloc king' to 'blocking' in the Introduction.
Extra space in a word is a typographical error.

Full text (46,902 chars)Analyzed: Jul 6, 2026

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