Histological and transcriptomic changes associated with matrix metalloproteinase 9 upregulation in rat esophageal...

Esophageal fistula (EF) is a serious complication associated with high morbidity, yet the local tissue changes that drive persistent fistula formation and failed healing remain poorly understood. This study established a surgically induced rat model of EF and characterized its histological and transcriptomic features, with particular attention to molecules potentially involved in inflammatory remodeling.

Fifty male Sprague-Dawley rats were randomly assigned to an EF group or a sham-operated control group (25 per group). EF was induced by cervical esophageal incision followed by indwelling tube placement. Model establishment was confirmed by gross inspection and methylene blue leakage testing. Histopathological changes were evaluated using hematoxylin-eosin (H&E), Masson's trichrome, and elastic van Gieson (EVG) staining, together with immunohistochemistry (IHC) for CD31, CD34, cytokeratin 5/6 (CK5/6), α-smooth muscle actin (α-SMA), and matrix metalloproteinase 9 (MMP9). Whole-transcriptome sequencing was performed on EF tissue and normal esophageal tissue from three rats per group, followed by differential expression and enrichment analyses. Selected candidate genes were further validated by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blotting.

The rat EF model was successfully established and showed full-thickness esophageal defects with persistent luminal communication. Histologically, EF tissue exhibited marked architectural disruption, dense inflammatory cell infiltration, muscular layer interruption, extracellular matrix (ECM) remodeling, and selective loss of elastic fibers. IHC demonstrated increased CD31- and CD34-positive vascular signals, expansion of CK5/6-positive basal-like epithelium, and accumulation of α-SMA-positive myofibroblasts in EF tissue. Transcriptomic profiling identified 1,550 differentially expressed genes (DEGs), including 1,128 upregulated and 422 downregulated genes. Enrichment analyses showed strong activation of immune-inflammatory and matrix-remodeling pathways, including cytokine-cytokine receptor interaction, chemokine signaling, tumor necrosis factor (TNF) signaling, interleukin-17 (IL-17) signaling, nuclear factor-kappa B (NF-κB) signaling, leukocyte transendothelial migration, and ECM-receptor interaction. RT-qPCR confirmed significant upregulation of C-X-C motif chemokine ligand 6 (CXCL6) and MMP9 at the messenger RNA (mRNA) level in EF tissue, and increased MMP9 protein expression was further verified by western blotting and IHC.

Rat EF is characterized by persistent inflammatory activation, abnormal stromal remodeling, selective elastic fiber loss, and incomplete structural restoration. MMP9 is consistently upregulated in this pathological microenvironment and may be associated with impaired healing in EF. This model provides a practical platform for future mechanistic studies of EF.