Development and Characterisation of a Minimally Invasive Fixed C7 Foraminal Compression Model of Cervical Radiculopathy with Transcriptomic Identification of P2X2

Abstract Objective To establish an SD rat model of cervical spondylotic radiculopathy (CSR) in which the implant is inserted through the C7 intervertebral foramen into the target region and fixed to the C7 spinous process; to evaluate the model’s suitability for this study through a combination of pain behavioural assessments, gait analysis, histomorphology of nerve roots and dorsal root ganglia (DRG), neuronal activation, detection of local inflammatory factors, and DRG transcriptomic analysis, to evaluate the model’s ability to reproduce the phenotypic manifestations of CSR pain, neural tissue damage and molecular alterations over the observation period of this study. Methods A total of 84 male SD rats were used in two independent experiments. Experiment 1 included 48 rats assigned to the Control, Sham, Model 7 d and Model 14 d groups (n = 12 per group) for model establishment and assessment of pain-related behaviour, gait, histopathology, inflammation and DRG transcriptomics. Experiment 2 included 36 rats assigned to the Control, Model 7 d, Model 14 d and Model 14 d + AF-353 groups (n = 9 per group) for molecular and pharmacological validation. The custom titanium hook was inserted into the C7 foraminal target region and fixed to the C7 spinous process. Mechanical and thermal nociceptive measures, spontaneous pain-related behaviours and CatWalk gait parameters were evaluated; HE staining, transmission electron microscopy, c-Fos and NeuN staining, ELISA and DRG transcriptomics were used to characterise tissue and molecular changes. P2rx2 mRNA and P2X2-related protein readouts were examined by RT-qPCR and Western blot. In Experiment 2, AF-353 was administered once intrathecally on postoperative day 14 at 30 µg in 10 µL; CatWalk analysis was performed 60 min after administration and terminal tissue collection at 90 min. Results Compared with the Control group, the Model 7 d and Model 14 d groups exhibited lower mechanical withdrawal thresholds and thermal withdrawal latencies, longer licking, biting and audible-vocalisation durations, more pain-related behavioural events, shorter stance time and stride length, and longer swing time. HE staining and transmission electron microscopy revealed structural and ultrastructural damage to the DRG and nerve root. Immunostaining showed increased c-Fos-related signals and altered NeuN-positive area, whereas ELISA showed increased DRG IL-1β, TNF-α and IL-6. DRG transcriptomics identified 580 differentially expressed genes and enrichment related to neuroactive-ligand signalling, membrane excitability, pain responses and Ca²⁺ transport. P2rx2 and Camk2a were upregulated, and P2rx2 occurred in the leading-edge subsets of both neuroactive-ligand signalling and calcium-ion transport gene sets. Following AF-353 intervention, P2rx2 mRNA, P2X2 protein, relative p-CaMKIIα and p-CREB abundance, and c-Fos-related readouts were lower, accompanied by changes in selected pain-related gait parameters. Conclusion This study established a CSR rat model based on local, sustained mechanical stimulation of the C7 intervertebral foramen, fixed via the C7 spinous process. During the 7–14-day observation period, this model exhibited radicular pain, forelimb gait abnormalities, damage to nerve roots and DRG tissue, neuronal activation and local inflammatory responses. DRG transcriptomic screening identified candidate nodes such as P2rx2 and Camk2a ; following AF-353 intervention, there was a decrease in P2rx2 mRNA, P2X2 protein, p-CaMKIIα, p-CREB and c-Fos levels, accompanied by partial improvements in gait. These results support the association between P2X-related purinergic signalling and Ca²⁺–CaMKIIα–CREB-related changes with DRG sensitisation following sustained nerve root stimulation; however, they do not constitute evidence of a P2X2-subtype-specific causal relationship. This model may serve as a candidate experimental platform for studies into the pathophysiological mechanisms and interventions for CSR.