CILP-2 expression in the intervertebral discs of patients with lumbar radiculopathy

Abstract

Background: Intervertebral disc (IVD) degeneration (IVDD) is one of the main causes of low back pain. One of the most important features of IVDD is the loss of extracellular matrix (ECM) with its structural components. Cartilage intermediate layer proteins (CILPs), minor glycoproteins residing in ECM, have been found to be increased in IVD as degeneration and aging progresses. The aim of the present study was to evaluate the expression of CILP-2 in the IVD of patients with lumbar radiculopathy.

Methods: The IVD samples were collected from 25 patients during spinal surgery (interlaminectomy, herniated disc removal). The control IVD samples were obtained from nine patients who underwent lateral corpectomies in the thoracic region. CILP-2 expression was detected by immunohistochemistry. The patients were divided into two groups - aged under or over 50 years. A standardized clinical examination with assessment of radicular signs and deficits was performed. Subjective disability and pain were assessed using the visual analogue scale and Oswestry Disability Index (ODI). The pre-operative MRI was graded for the degree of IVD degeneration by Pfirrmann grading system. IVD samples obtained during operations were subjected to the standardized histopathological analysis applying modified Boos classification. The data were analysed by t-test, Mann-Whitney U-test, and Spearman correlation test.

Results: Both histopathology scores and Pfirrmann grades did not differ between patients' groups. Also, no correlations were found between histopathology and Pfirrmann grades, neither were any differences seen when correlating both grades to ODI, back pain or leg pain scores. CILP-2 staining was noted in all studied samples, notably strong staining was seen around large cell clusters. However, no differences in CILP-2 staining were seen between the age groups of patients. No correlations were found between CILP-2 staining and Pfirrmann grades. Grading of CILP-2 immunostaining in nine control patient samples resulted in significantly lower values. The difference is statistically significant (P = 0.002) compared to CILP-2 staining scores of all 25 patients' samples.

Conclusion: In this study, we detected increased CILP-2 expression in the human IVD as compared to the control group patients. CILP-2 can be a possible IVDD marker; however, as knowledge about the role of CILP-2 is limited, further studies are required.

Keywords: Cartilage intermediate layer protein; Immunohistochemistry; Intervertebral disc degeneration; Radiculopathy.

Fig. 1

The grading of patients’ groups based on subjectively evaluated radicular pain in the back or in the legs (grading from 1–10), according to radiological disc degeneration evaluated by modified Pfirrmann grading system based on measurement of the signal intensity on T2-weighted MR images (grading ranging from 3 to 7), according to pathohistological evaluation of IVD samples (grades ranging from 0–12 based on estimations of cell density, structural alterations and mucoid degeneration) and according to the assessment of the percentage of the CILP-2 stained area. GI – group of patients younger than 50 years, GII – group of patients older than 50 years. Box-whiskers plot with 5th–95th percentiles. *P = 0.005, Mann-Whitney U-test

Fig. 2

Representative micrographs of IVD samples with large clusters of cells. A (specimen of GI group), note large clusters of cells (white arrows), B (specimen of GI group), large cluster of cells surrounded by intensively stained acidic mucopolysaccharide matrix (black arrowhead). A – safranin O, B – alcian blue-PAS

Fig. 3

Immunohistochemical staining of CILP-2 in IVD samples. A (GII specimen) and B (GI specimen). Note CILP-2 staining in the extracellular matrix with particularly strong pericellular expression (white arrows). C (GI specimen). Characteristically strong CILP-2 staining around the large cluster of cells (black arrowhead). D and E (GI specimen). Note strong CILP-2 staining in the vicinity of cells (white arrows), rectangle in D presented at higher magnification in E. F (control group specimen). Note weak CILP-2 staining in the extracellular matrix, white arrows indicate positively stained cells. DAB + toluidine blue

Fig. 4

Estimations of CILP-2 immunohistochemical staining by assessing the percentage of the stained area. All lumbar discectomy patients (n = 25) vs. control group patients (n = 9), significant difference by the Mann-Whitney U-test, *P = 0.002. Box-whiskers plot with 5th–95th percentiles