Elastogenic Protein Expression of a Highly Elastic Murine Spinal Ligament: The Ligamentum Flavum

Spinal ligaments, such as the ligamentum flavum (LF), are prone to degeneration and iatrogenic injury that can lead to back pain and nerve dysfunction. Repair and regeneration strategies for these tissues are lacking, perhaps due to limited understanding of spinal ligament formation, the elaboration of its elastic fibers, maturation and homeostasis. Using immunohistochemistry and histology, we investigated murine LF elastogenesis and tissue formation from embryonic to mature postnatal stages. We characterized the spatiotemporal distribution of the key elastogenic proteins tropoelastin, fibrillin-1, fibulin-4 and lysyl oxidase. We found that elastogenesis begins in utero with the microfibril constituent fibrillin-1 staining intensely just before birth. Elastic fibers were first detected histologically at postnatal day (P) 7, the earliest stage at which tropoelastin and fibulin-4 stained intensely. From P7 to P28, elastic fibers grew in diameter and became straighter along the axis. The growth of elastic fibers coincided with intense staining of tropoelastin and fibulin-4 staining, possibly supporting a chaperone role for fibulin-4. These expression patterns correlated with reported skeletal and behavioral changes during murine development. This immunohistochemical characterization of elastogenesis of the LF will be useful for future studies investigating mechanisms for elastogenesis and developing new strategies for treatment or regeneration of spinal ligaments and other highly elastic tissues

Timeline summarizing elastogenesis in LF during development, maturation and aging.

<p>Timeline summarizing elastogenic protein expression in the LF; stages/ages are in days unless otherwise indicated. Not drawn to scale.</p

Fibulin-4 immunostaining of LF during development, maturation and aging.

<p>Fibulin-4 staining of LF (arrows point just inside edge of tissue) was high in intensity from P7 through P21, and was less intense at all other ages studied. Scale bar: 50 µm.</p

Fibrillin-1 immunostaining of LF during development, maturation and aging.

<p>Fibrillin-1 staining of LF (arrows point just inside edge of tissue) was most intense at E17. Staining intensity was high from E16 through P0 and at P14, but was less intense at P28 and older ages. Scale bar: 50 µm.</p

Tropoelastin immunostaining of LF during development, maturation and aging.

<p>Tropoelastin staining of LF (arrows point just inside edge of tissue) increased in intensity significantly first at P7 and remained high until P28. Staining intensity gradually decreased after P28 to a consistently low coloration that persisted throughout adulthood. Scale bar: 50 µm.</p

LOX staining of LF during development, maturation and aging.

<p>LOX staining of LF (arrows point just inside edge of tissue) was intermittently detectable through the stages studied, with higher staining intensities detected at E16, E18, and P7 through P21. Scale bar: 50 µm.</p

H&E staining and cellularity results of LF.

<p><i>Top Three Rows:</i> Representative images of H&E-stained LF (arrows point just inside edge of tissue) from all stages characterized. Scale bar: 50 µm. <i>Bottom Panel:</i> Results of cell nuclei quantification in LF sections from E16 to P2-yrs embryos and mice. Cellularity of the LF shows a significant decrease at P14, and appeared to be lowest at P2-yrs. Mean ± standard deviations, <i>n</i> = 3. *<i>P</i><0.05 versus P0.</p

Determining cellularity of LF.

<p>Three representative 1000 µm² areas of the LF were selected for cell counting at approximately equidistant regions along the length of the ligament. Counts were averaged for mice at a given stage for comparison (P56 shown). Scale bar: 200 µm.</p