The paradoxical relationship between ligamentum flavum hypertrophy and developmental lumbar spinal stenosis

Measurements of spinal canal diameter and LF thickness were extracted from preoperative T1- and T2-weighted axial MRI scans. A 3T MRI was obtained of all subjects. The field of view was 18 x 18 cm for axial scans and 28 x 28 cm for sagittal scans. Slice thickness was 4 mm for both scans, and slice spacing was 0 mm for axial scans and 0.5 mm for sagittal scans. Imaging matrix was 288 x 192 for axial scans and 512 x 224 for sagittal scans. The repetition time was 700 to 800 ms and 4000 to 6000 ms for T1 and T2, respectively. The echo time was 8 to 10 ms and 80 to 100 ms for T1 and T2, respectively. There were 11 slices per vertebral level and parallel slices were made according to the disc and pedicle levels. A spine surgeon blinded to the patient details performed all measurements using Centricity Enterprise Web V3.0 software (GE Medical Systems, 2006). All canal diameter measurements were performed first for all patients and then all LF were measured after the order of images measured were randomly selected. Spinal canal diameters at L1, L2, L3, L4, L5 and S1 were assessed at the vertebral level (Fig. 1). Due to its anatomical location, LF thickness was measured at L1-L2, L2-L3, L3-L4, L4-L5 and L5-S1 intervertebral disc levels. The AP spinal canal diameter (Fig. 1) was measured using T1-weighted axial MRI, at the cut where the entire bony canal ring could be seen and with the thickest pedicle width [9]. The LF was measured (Fig. 2) at the midpoint of the facet joint level using T2-weighted axial MRI at the cut with the thickest LF measured.

As disc degeneration could be a possible cause for spinal canal narrowing and might be a confounder to the lumbar spinal stenosis assessment, it was used to stratify our results. Disc degeneration was characterized by a reduction in disc height. Thus, the disc height was assessed on T2-weighted MRI sagittal images to correlate with other imaging findings. Disc height was measured from the middle of the intervertebral disc (from the thickest cut) perpendicular to the endplate, inclusively from L1-2 to L5-S1 at each level. Any anterior disc bulging was also noted.

As this study aimed to investigate the characteristics of LF corresponding to canal size, it was necessary to differentiate patients based on the origin of neural compression and whether there was an existing narrowed canal. Based on the phenotypical definitions of DSS by Cheung et al., [10] patients were classified into 3 groups: DSS with critical stenosis (Group 1), DSS with relative stenosis (Group 2), and non-DSS (Group 3). Measurements were based on the AP bony spinal canal diameter at the vertebral level. Critical stenosis values were defined as: L4 < 14 mm, L5 < 14 mm, and S1 < 12 mm. DSS was defined as: L1 < 20 mm, L2 < 19 mm, L3 < 19 mm, L4 < 17 mm, L5 < 16 mm, and at S1 < 16 mm.

Excised LF was obtained during surgery at each operated level and was separated into left and right sides. Excised LF was fixed in 4 % (weight/volume) paraformaldehyde in phosphate-buffered saline (pH 7.4) immediately upon removal, then stored at 4 °C for 48 h before processing, and embedded in paraffin wax blocks longitudinally or in cross-section. Specimens were prepared in 5 μm sagittal sections according to the excised level and sides using a microtome.

Masson’s trichrome staining was performed for each LF specimen using the protocol by Carson [20], with the main staining solutions being Weigert’s Haematoxylin, Biebrich Scarlet-Acid Fuchsin and Aniline Blue working solutions. Nuclei, collagen fibres and keratin/elastin/muscle fibres were represented by black, blue and red colors, respectively. The slides were viewed under Nikon Eclipse 80i microscope and microscopic images were captured by computer imaging software (NIS-Elements F4.30.01 64-bit, Nikon, Japan). Each LF specimen was assessed for the degree of fibrosis, which was represented by the area occupied by collagen fibers out of the entire cut section. This was calculated using ImageJ 1.48d (RSB, NIMH, Maryland, USA), and was expressed in percentages. For the ease of comparison, grading system (Grade 0–4) defined by Sairyo et al. [21] was also used to indicate histological findings: Grade 0 represents normal tissue with no fibrosis, Grade 1 represents fibrosis less than or equal to 25 % of the entire area, Grades 2 and 3 represent fibrosis occurring between 25–50 % and 50–75 % of the entire area, respectively, and Grade 4 represents fibrosis of more than or equal to 75 % of the entire area (Fig. 3).

In Group 3, LF thickness was strongly correlated to the spinal canal diameter (Right: r = 0.98, p ≤ 0.001; Left: r = 0.92, p ≤ 0.01), and a weaker relationship for Group 2 at L5-S1 only (r = 0.75, p < 0.005). Group 1 failed to demonstrate any significant correlation of LF thickness and canal diameter at all, despite its pre-existing narrower canal space.

There were 55 operated intervertebral levels, giving rise to a total of 110 LF specimens collected. With comparable mean LF thickness according to individual intervertebral levels as expressed in Table 2, a significant negative correlation of LF thickness and fibrotic grades was found in both Groups 1 and 2 (Table 3).

The LF thickness was also found to have an inverse relationship with the area of fibrosis (%) in Group 1 (Table 3). On the contrary, Group 3 had a significant positive correlation of LF thickness and fibrosis area. These relationships were even stronger when analyzing particular intervertebral levels, especially at L4-5 for Groups 1 and 2 demonstrating significant negative correlations, and at L5-S1 where Group 2 demonstrated an inverse relationship and Group 3 exhibited a very strong positive relationship (Table 4).