2.2 | LF thickness measurement and fibrosis evaluation
As previously stated (Sun et al., 2018), the LF thickness was assessed at the facet joint level on the T2-weighted MRI from all the 42 patients. The measurement was performed on the Picture Archiving and Communication Systems (PACS) software (Nanjing Medical University, Nanjing, China). The value was measured three times for each patient by one experienced spine surgeon and the average value was considered as the LF thickness (Fig. 1A ).
All the LF samples collected for this study were only harvested from the dorsal layer of LF. LF samples were fixed in 4% paraformaldehyde phosphate buffer solution for 48 h and subsequently embedded in paraffin in preparation for histopathological analysis. The samples were sliced along the coronal plane for each specimen at the same level by using a paraffin microtome. After dewaxing, the sections were stained using H&E staining, Masson’s trichrome(MT) staining and elastica van Gieson (EVG) kits (JianChen, Nanjing, China). H&E and EVG staining was used to evaluate the morphology and structure of the LF. Moreover, the degree of LF fibrosis was assessed according to the results of MT staining. The fibrotic area was quantified through Image Pro Plus 6.0 (China). As previously described (Sun et al., 2017), the grade of LF fibrosis score was classified as follows: Grade 0 indicated collagen less than 20% of the entire area; Grade 1, ≤25% of the entire area; Grade 2, between 25% and 50%; Grade 3, between 50% and 75%; and Grade 4, over 75% of the whole area of the sample.