We will conduct a systematic review of preclinical animal studies, according to the guidelines recommended in PRISMA-P (Preferred Reporting Items for Systematic Review and Meta-Analysis Protocols) [23]. A completed PRISMA-P checklist can be found as Multimedia Appendix 1. In preparing this protocol, we have also adopted an animal systematic review protocol format developed by de Vries et al [24], which was designed specifically for review of animal studies. Any changes or amendments made to the protocol will be recorded in PROSPERO as minor amendments or major revisions, following their procedures, with each update including the date, version number, and a summary of the changes. Any deviations from the registered protocol will also be justified and documented in the final manuscript.

A literature search will be conducted in MEDLINE (Ovid), Embase, and Web of Science by using terms related to the research question, including but limited to “cutaneous neurogenic inflammation” and “Evans Blue dye extravasation.” The initial search strategy was jointly developed by two investigators (EY and PW) and a medical librarian/information scientist (AC). It was then reviewed and refined based on suggestions and feedback from other co-investigators. The search will be limited to English-language publications. No limitation will be placed on the publication date range. In addition to electronic database search, we will hand search references of the included studies to identify additional relevant studies. The full search strategies for all databases can be found in Table 1. The search will be rerun prior to data synthesis to capture newly published studies, and updated search dates and strategies will be documented in the supplementary materials and reported in the final manuscript to ensure transparency and reproducibility.

Of note, we found that some of the preclinical studies on this topic were conducted by acupuncture researchers aiming to compare the locations and distributions of cutaneous neurogenic inflammatory spots with that of acupuncture points, the specific points on the body surface that are stimulated during acupuncture treatment [15-20]. Thus, we also included terms related to “acupuncture points” to be more comprehensive in our search strategy (Table 1).

A summary of the inclusion and exclusion criteria is provided in Textbox 1. Briefly, this review will include preclinical animal studies, investigating experimentally induced models of acute or chronic visceral diseases. Studies involving humans, in vitro, or ex vivo experiments will be excluded. Studies using EB dye or other validated markers of plasma extravasation will be considered for inclusion. EB is an azo dye that binds strongly to blood albumin, and its leakage into the surrounding tissue can be visualized to assess the extent and distribution of cutaneous neurogenic inflammation [25,26]. Only studies with a control or baseline comparison and sufficient methodological detail for data extraction will be included.

Two investigators (EY and DMV) will independently screen all the studies resulting from the literature search to include relevant studies that meet the predefined inclusion and exclusion criteria. Screening will first be conducted based on titles and abstracts. Studies that do not meet the eligibility criteria will be excluded at this stage. The remaining studies will be screened by retrieving and examining the full texts for eligibility. Interrater agreement will be assessed using Cohen κ. Any disagreements between reviewers will first be discussed to reach a consensus; if unresolved, they will be adjudicated by a third investigator (PW).

Duplicate publications and overlapping datasets will be identified by cross-checking study details (eg, author names, animal models, experimental methods). When multiple reports describe the same dataset, the most comprehensive or recent publication will be included, and additional information from other reports will be extracted as appropriate to avoid duplication.

Two investigators (EY and DMV) will independently extract data from all included studies, using a predefined template (Multimedia Appendix 2). Data extraction will first be piloted on a subset of studies to ensure consistency and completeness between reviewers. Any discrepancies during data extraction will be resolved through discussion and consensus; if unresolved, they will be adjudicated by a third investigator (PW).

Briefly, the data extraction table includes the following: (1) publication details (eg, title, authors, publication year); (2) study population (eg, details on animal species and strains, sample size, and other characteristics of the animals such as sex and age); (3) visceral disease model, including the details of the methodology used to induce the disease model (eg, name and dosage of chemical irritant used to induce the disease model); (4) comparison or control group (eg, use of saline); (5) outcomes measures (eg, comparisons of EB dye extravasation in disease and control groups in terms of distribution and numbers of blue dots), including the details of the methodology (eg, dosage and route of EB dye administration); (6) outcomes and methods relating to pain sensitivity assessment of blue dots; (7) any other relevant methods and results from included studies, which provide additional insights about the neurocircuitry of referred visceral pain as well as other pathophysiological features of the skin in visceral diseases; and (8) if applicable, pertinent information about acupuncture point(s) (eg, names of acupuncture points in close proximity to blue dots).

The primary outcome of this review will be the extent of cutaneous neurogenic inflammation in response to experimental induction of visceral disease, as assessed by the distribution and extent of EB dye extravasation. Secondary outcomes will include pain sensitivity assessments as well as additional measures of neurogenic inflammation such as immunohistochemistry for neuropeptides (eg, substance P, calcitonin gene-related peptide), if available. Acupuncture point-related data will also be considered secondary and extracted only when directly relevant to the main mechanistic question.

The included studies will be independently assessed for their methodological quality and risk of bias by two investigators (EY and DM-V). Given that this review involves preclinical animal studies, we will adopt the Risk of Bias (RoB) tool from the Systematic Review Centre for Laboratory Animal Experimentation (SYRCLE), which has been developed to address the specific challenges and complexities associated with assessing the rigor of evidence derived from animal research [24,27]. Briefly, SYRCLE’s RoB tool includes 10 entries related to 6 types of bias: selection bias, performance bias, detection bias, attrition bias, reporting bias, and other biases. This tool is designed to be applicable across different animal species and experimental models. Using this RoB tool, we will identify any potential limitations in the design, conduct, and reporting of included studies and determine the overall strength of evidence regarding cutaneous neurogenic inflammation in visceral diseases. Both reviewers have previously been trained in the use of the SYRCLE risk of bias tool and will independently assess the included studies and then discuss their evaluations to reach consensus. Any disagreements will be resolved through discussion and consensus or review; if unresolved, they will be adjudicated by a third investigator (PW).

The findings of individual studies included in this review will be synthesized to identify overall patterns and draw conclusions relevant to the research question, taking into account the methodological quality of the included studies. For outcomes related to the distribution and extent of EB dye extravasation, a descriptive synthesis will be performed to summarize key trends across models, including species, visceral organ involved, and induction method. Given the anticipated heterogeneity in study design, disease induction, and outcome quantification, a meta-analysis will not be planned at this stage. Instead, findings will be reported narratively, highlighting consistent observations and methodological differences among studies. If sufficient homogeneity and quantitative data become available, a meta-analysis may be considered in future analyses.