Fig. 7

Sorafenib can inhibit ossification and improve neurological symptoms in Ligament Spontaneous Ossification (LSO) model. a Representative 3D reconstructed Micro-CT images of the cervical spine. Following 14 weeks of intragastric administration of sorafenib or vehicle, the cervical spine of the mice was scanned and reconstructed in transverse (upper) and sagittal (lower) positions. This evaluation aimed to assess the occupation of ossified ligament tissue within the spinal canal of the LSO model. Ossified ligaments are indicated by white arrows and dotted boxes. Scale bars = 2 mm. b Histograms displaying the occupancy ratio of the spinal canal and the Ossification (OS) index of the cervical spine. Data is presented as means ± SD (n = 6). Statistical significance was assessed using Student’s t-test (***P < 0.001). c Von Frey assay (left) and thermal hyperalgesia test (right) were conducted on the right hind paw of mice treated with vehicle or 50 mg/kg sorafenib at 18 weeks. Error bars represent mean values ± SD (n = 6). Statistical significance was determined using Student’s t-test (***P < 0.001). d, e Representative safranin O and Fast Green (SOFG) staining images of the C2 (upper) and C5 (lower) cervical spine sections treated with or without sorafenib, accompanied by the quantification of the cartilage area ratio within the ossified tissue. Scale bars =100 μm, n = 6. Statistical significance was determined using Student’s t-test (***P < 0.001). f–i Immunostaining of cervical spine sections from each group with indicated antibodies, along with quantitative analysis. Scale bar = 40 μm. Error bars represent mean values ± SD (n = 6). Statistical significance was assessed using Student’s t-test (**P < 0.01, ***P < 0.001). j, k Representative immunofluorescence images illustrating EMCN (green) and CD31 (red) staining within the ossified ligament of mice treated with vehicle or sorafenib, accompanied by corresponding quantitative analysis. Scale bar = 50 μm. Error bars represent mean values ± SD (n = 6). Statistical significance was determined using Student’s t-test (***P < 0.01). All experiments were conducted with three independent biological replicates