Specimens Collection
The research program was approved by the Institutional Review Committee of Tianjin Union Medical Center, and all procedures were performed according to the Declaration of Helsinki. All patients received written informed consent before operation. The LF was collected during surgery after obtaining the informed consent from patients. This protocol was approved by the institutional ethic boards of Tianjin Union Medical Center. Thirty LF specimens were obtained from 30 patients who had undergone decompressive laminectomy for neurogenic claudication to SLSS and DLSS. Thirty LF specimens were obtained from 30 patients in LDH patients during surgery. The baseline data between each group has no significant difference (p >0.05, Table 1). All the patients had been unresponsive to conservative measures for at least 3 months. None of these patients receive selective nerve-root blocks. patients who had isthmic and degenerative spondylolisthesis, scoliosis, or fractures were excluded from this study. We collected the entire layer of the LF and removed all the epidural fat from the LF specimens. Each specimen was fixed in 4% neutral formalin, decalcified with 20% ethylenediaminetetraacetic acid (EDTA) for four to six weeks and then embedded in paraffin for histologic and immunohistochemical analysis.
MRI Measurement
MRI examination was performed before operation. MRI T1 phase cross-section measurement was performed by hospital PACS system in Tianjin Union Medical Center. The thickness of the LF was compared in each group as proposed by Fukuyama[16] .The thickness of the LF was measured from the mid-point of the LF to the ventral side of the inner rim. The lumbar spinal canal oblique diameter is measured from the midpoint of the dorsal side of the ligamentum flavum to the midpoint of the posterior margin of the vertebral body. The relative thickness (RT) (%) of LF is calculated as the percentage of LF thickness compared to lumbar spinal canal oblique diameter. 3 independent measurements from 3 surgeons were averaged to determine the RT of an individual sample.
Histologic Analysis for Elastin Degradation and Fibrosis of the LF
Two consecutive sections (4 μm thickness) were obtained and stained with HE and VVG stain, respectively. HE stain was used to characterize the LF collagen deposition and VVG stain was used to characterize the elastic fiber. Histologic analysis was independently performed by 3 pathologists on 10 randomly selected, high power fields (× 400) images of each sample.
The HE stained slides were independently evaluated and graded according to LF elastin degradation. Grade 0 indicates normal tissue which shows no elastin degradation region. Grade 1 indicates that elastin degradation is < 25% of the entire area. Grade 2 indicates between 25% and 50% elastin degradation. grade 3 indicates a 50% to 75% elastin degradation and grade 4 indicates > 75% elastin degradation.
Scores were assigned to each VVG stained slide based on the presence and morphology of the LF elastic tissue. The following grading criteria were used: 0, normal; 1, short fragmented elastic fibers; 2, intermediate between 1 and 3; 3, fibrillar elastic fibers; 4, intermediate between 3 and 5; and 5, absent or nearly absent.
All HE and VVG stained slides were viewed using an Olympus BX50 light microscope (Olympus Corp), and digital images were taken at ×200 magnification with an Olympus DP20 microscope camera. The image files were saved as high-resolution tag image file format files. Images were captured from dural and dorsal aspects regions of the LF.
Immunohistochemical Analysis for the Localization of P16 and S100
The LF specimens were fixed in 10% neutral formalin and embedded in paraffin. 5 μm thick sections were collected, dewaxed in xylene, and rehydrated in graded ethanol solutions. Sections were then incubated with purified mouse monoclonal antibody specific to P16 (Maixin Biotechnology, Fuzhou, China, 1:100) or purified mouse monoclonal antibody specific to S100 (Maixin Biotechnology, Fuzhou, China, 1:100). The positive controls were also performed according to the manufacturer’s recommendation. A routine immunoperoxidase staining technique using 3,3-diaminobenzidine tetrahydrochloride was performed.
Statistical Analysis
The results of the absolute and relative thickness, and the histological ratings of HE and VVG stained slides of the three groups were compared using one-way ANOVA. We determined the relationships between the thickness and the histological ratings of HE- and VVG-stained slides using Pearson’s correlation coefficient test. Data are shown as mean ± SD, and a p value less than 0.05 was used to determine statistical significance. The IBM SPSS Statistics version 21.0 software (IBM, New York, NY, US) was used for all analysis.