Development of a Novel Murine Model for Reversible Pulpitis and Identification of Biomarkers: Exploring a Neurodegenerative Hypothesis

Abstract

Accurate diagnosis of pulpal disease remains a major challenge in endodontics, particularly when attempting to distinguish between reversible and irreversible pulpitis. While current clinical tests are effective in determining pulp vitality, they lack the specificity required to assess the true histological status of the tissue. This diagnostic limitation has become increasingly problematic with the advent of vital pulp therapies, which rely on the preservation of pulpal vitality and therefore require precise case selection. The aims of this study were to develop a reproducible murine model to define reversible and irreversible pulpitis based on histological criteria and to identify neurodegenerative biomarkers associated with pulpal injury that could serve as objective diagnostic tools. A total of twelve C57BL/6j mice were divided into four groups based on pulp exposure time: 0 hours, 24 hours, 72 hours, and 7 days. Following mechanical pulp exposure, teeth were left open to the oral environment for the designated period before being capped with Biodentine™ and allowed to heal for 14 days. Histological analyses were performed using Hematoxylin and Eosin staining to assess tissue structure and necrosis, immunohistochemistry targeting β-tubulin III to evaluate neuronal integrity, and fluorescence in situ hybridization to quantify bacterial invasion. Based on these analyses, the 0-hour and 24-hour exposure groups were identified as representative models of reversible and irreversible pulpitis, respectively. Quantitative histological assessment demonstrated a significant increase in necrotic pulpal tissue from 35% at 0 hours to 85% at 24 hours, confirming a clear threshold between reversible and irreversible injury. Immunohistochemical analysis revealed preservation of neuronal structure at 0 hours, while near-complete loss of β-tubulin III signal was observed at 24 hours, indicating extensive neuronal degeneration. Fluorescence in situ hybridization showed progressive bacterial invasion over time, although the extent of tissue damage appeared more closely associated with host inflammatory responses than with bacterial load alone. To explore the potential for objective, non-invasive diagnostics, gingival crevicular fluid, saliva, and pulp tissue samples were analyzed using the Olink Target 48 proteomic platform, focusing on neurodegenerative biomarkers. Results identified several proteins associated with neuronal injury and oxidative stress, including nerve growth factor (NGF), neurofilament light chain (NEFL), and glutaredoxin (GLRX). NGF was significantly upregulated in reversible pulpitis, while NEFL and GLRX showed increased expression in irreversible pulpitis, suggesting their potential utility as stage-specific biomarkers. Additional markers, including SYT1 and SCG2, were significantly downregulated in irreversible cases, reflecting loss of neuronal function. This study demonstrates that pulpal disease progression is closely associated with neuronal degeneration, supporting a neurodegenerative component to pulpitis. The murine model developed herein provides a reliable framework for studying pulpal pathology over time, while the identification of neuron-specific biomarkers highlights a promising avenue for improving diagnostic accuracy. Ultimately, the integration of biomarker-based diagnostics could enhance clinical decision-making, allowing for more predictable selection of vital pulp therapies and improved patient outcomes. Further research is warranted to validate these findings in human subjects and to explore their clinical applicability.