Normal aging in human lumbar discs: An ultrastructural comparison

Abstract

The normal aging of the extracellular matrix and collagen content of the human lumbar intervertebral disc (IVD) remains relatively unknown despite vast amounts of basic science research, partly because of the use of inadequate surrogates for a truly normal, human IVD. Our objective in this study was to describe and compare the morphology and ultrastructure of lumbar IVDs in 2 groups of young (G1-<35 years) and elderly (G2->65 years). Thirty L4-5 and L5-S1 discs per group were obtained during autopsies of presumably-asymptomatic individuals and analyzed with magnetic resonance imaging (MRI), a morphological grading scale, light microscopy, scanning electron microscopy (SEM) and immunohistochemistry (IHC) for collagen types I, II, III, IV, V, VI, IX and X. As expected, a mild to moderate degree of degeneration was present in G1 discs and significantly more advanced in G2. The extracellular matrix of G2 discs was significantly more compact with an increase of cartilaginous features such as large chondrocyte clusters. Elastic fibers were abundant in G1 specimens and their presence correlated more with age than with degeneration grade, being very rare in G2. SEM demonstrated persistence of basic structural characteristics such as denser lamellae with Sharpey-type insertions into the endplates despite advanced age or degeneration grades. Immunohistochemistry revealed type II collagen to be the most abundant type followed by collagen IV. All collagen types were detected in every disc sector except for type X collagen. Statistical analysis demonstrated a general decrease in collagen expression from G1 to G2 with an annular- and another nuclear-specific pattern. These results suggest modifications of IVD morphology do not differ between the anterior or posterior annulus but are more advanced or happen earlier in the posterior areas of the disc. This study finally describes the process of extracellular matrix modification during disc degeneration in an unselected, general population and demonstrates it is similar to the same process in the cervical spine as published previously.

Fig 1. MR and corresponding mid-sagittal views of lumbar vertebral blocks.

A typical G1 specimen (A, B) is shown with incipient degenerative findings. Advanced degenerative findings in G1 specimens were almost always posterior annular tears as shown in the L5-S1 disc of this other specimen (C, D–Okada 3, Thompson 4 for L5-S1). Forty percent of all G2 discs exhibited structural failure as shown in E and F (Okada 6 and Thompson 5).

Fig 2. Distribution of Okada (n = 10) and Thompson (n = 30) scores per group.

Bars, median and interquartile range. Mann-Whitney test.

Fig 3. Light microscopy images.

Alternating diagonal and longitudinal lamellae are visible in the aAF and predominantly longitudinal in the pAF. Sharpey-type insertion of these fibers into the endplate is visualized in G1 (asterisks) and G2. The loose fibrocartilaginous phenotype of the G1 AF is substituted for a cartilaginous one in G2 with dense extracellular matrix and chondrocyte clusters. HE, hematoxylin & eosin. SR, Sirius Red. “C” designates a coronally-oriented section, Th notes Thompson grade, arrow indicates the nearest endplate.

Fig 4. Elastic fiber stains in G1 and G2 lumbar discs.

The presence of elastic fibers was more related to group (age) than to degeneration grade; even Th4-5 G1 specimens had prominent fibers in the AF while mildly-degenerated G2 discs (Th2) uniformly lacked them. The single G2 specimen is shown with few elastic fibers in the pAF (arrows). V, Verhoeff; W, Weigert; Th1-5, Thompson grade.

Fig 5. Scanning electron microscopy.

Typical AF arrangement seen in 5A, with 1–2μm interlamellar space. Endplate insertion of AF fibers is visible in 5G and 5K and not present in the NP (5C and 5I). A cartilaginous phenotype predominates in G2 specimens, with a denser matrix in both the AF and NP. Lamellae are compact and no space is visualized even at higher magnification (5H and 5L). pAF tears are demonstrated (**) in G1 (5E) and a G2 (5K).

Fig 6. Stained area (%) per collagen type.

Vertical arrows indicate p < .05 in G1 vs. G2 comparison. Black asterisks indicate p < .05 in the aAF vs. pAF comparison and are located in the frame with higher collagen reactivity. NS, not significant. Error bars, standard error of the mean.