Inquisition of the Human Intervertebral Disk Histopathology with the Use of Two Histological Staining Methods

Abstract

IntroductionChronic low back pain (LBP) affects over 10% of the adult population resulting in profound decreases in the quality of life. The intervertebral disk (IVD) is prone to degeneration which may contribute toLBP. To prevent disk degeneration-related LBP, better understanding of disk structure and the degenerative process is needed to understand the underlying mechanisms. Standard Hematoxylin and Eosin (H&E) staining, alone or in combination with other dyes, have been used to assess the normal biology and degeneration of IVDs. Recently, the multichromatic FAST (Alcian blue, Safranin-O, Fast green and Tartrazine) staining method has been developed (Leung V.Y.L. et al., 2009). This method provides additional information unavailable through conventional methods such as differentiation of the structures within the disk and identification of degenerative matrix remodeling through changes in the staining pattern. Although the FAST method has proved helpful in animal models to assess alteration of glycosaminoglycan content, its usefulness to assess human disk histopathology remains unknown.Materials and MethodsHuman disks were obtained (a) surgically from chronic LBP patients with moderate-severe disk degeneration or (b) postmortem from transplant donors. Disks were cut into quadrants, fixed in 4% paraformaldehyde with 14% (v/v) saturated picric acid for 3 days, embedded in OCT and stored at −80°C. Tissue samples were cut on a cryostat and transverse sections of 14 um and 20 um were obtained for H&E and FAST staining, respectively. Slides were coverslipped with DPX and scanned using a Zeiss MIRAX Scan Digital Slide Scanner for further examination.ResultsThe FAST protocol creates high-resolution and polychromatic images of the general disk structure, and reveals the overall organization of the disk. The transition from the nucleus pulposus (NP) to the inner annulus fibrosus (IAF) and the outer annulus fibrosus (OAF) was clearly visible through color boundaries. Signs of degeneration could be observed with the FAST staining method in both IVDs obtained postmortem with no history of LBP and from chronic LBP patients. For example, FAST staining uncovered the loss of a clear boundary between the NP and the IAF that was not visible with H&E. These data suggest that relevant pathological details might be missed if assessing histological changes with the use of H&E only.ConclusionThe combined use of H&E and FAST methods provides a more complete picture of the anatomy and physiology of human IVDs. The FAST profile distinguishes IVD compartments and shows matrix remodeling events within the disk, while H&E provides complementary information regarding the cellular content of the tissue. These two staining methods complement each other to give a clearer differentiation of IVD sub-regions of both normal and degenerated disk architecture.I confirm having declared any potential conflict of interest for all authors listed on this abstractYesDisclosure of InterestNone declaredLeung V.Y.L. et al. Journal of Histochemistry and Cytochemistry 2009;57:249