Innervation patterns of PGP 9.5-positive nerve fibers within the human lumbar vertebra

Abstract

Intervertebral disc injury or degeneration is a common cause of low back pain, and yet the specific source of pain remains ambiguous in many cases. Previous research indicates that the central vertebral endplate is highly innervated and can elicit pain responses to pressure. In effort to trace the origin of nerves located at the endplate, we used protein gene product 9.5 (PGP 9.5) to stain neurofibers and then quantified the spatial pattern of nerve distribution within a human L4 lumbar vertebra. The majority of nerves were adjacent to blood vessel walls, and consequently the nerve distribution closely resembled previously established vascularity patterns. We observed that the majority of nerves enter the vertebral body posteriorly, via the basivertebral foramen, and cluster in the vertebral center. These nerves follow the course of the nutrient artery, which enters the vertebral body through the basivertebral foramen, then branches toward the superior and inferior endplates. Our observations support the notion that nerves found at the central endplate could originate from sinuvertebral nerves accompanying the nutrient artery into the vertebral body. We also stained neighboring histological sections with calcitonin gene-related protein and noted significant co-localization with PGP 9.5, substantiating a nociceptive role for the nerves constituting our distribution pattern.

Fig. 1

(A) An image composite of an example coronal section. A small area is selected within the foramen and magnified to demonstrate the marking process first showing unmarked nerves stained dark brown with PGP9.5, then having been marked, and lastly an image of the nerves only. (B) The orientation of nine red-outlined regions of interest (ROI). The data from these nine ROI are then presented along the y-axis and organized for each of the 34 sections along the x-axis. These graphs generate a sagittal view of the nerves distribution throughout the vertebral body.

Fig. 2

(A) A blood vessel stained with calcitonin gene-related protein. (B) The same blood vessel but 5 μm away and stained with protein gene product 9.5 (PGP 9.5). (C) A blood vessel stained with NF200. (D) The same blood vessel but 9 μm away and stained with PGP 9.5.

Fig. 3

Top composite image shows where the following images A through D are located along a coronal section of the posterior vertebral body. (A) The large vessels and nerves located in the basivertebral foramen. (B) A nerve unassociated with a blood vessel above and a typically innervated blood vessel below. (C) A very thin nerve moving diagonally and located near the superior endplate. (D) Another typically innervated blood vessel.

Fig. 4

For each graph, nerve data from each of the nine regions of interest (ROI) is oriented along the y-axis. The x-axis organizes the 34 sections from anterior to posterior with data from each ROI composing a complete sagittal view of the L4 interior. L4 Nerve Area depicts the quantity of nerve (mm²) in each ROI among the 34 sections. L4 Nerve Count shows the number of individual nerves in each ROI among the 34 sections.

Fig. 5

(A) An adaptation from Crock & Yoshizawa (1976), demonstrating the vascularity seen across a sagittal cross-section of a lumbar vertebra. (B) A nerve density graph from our L4 vertebra correlating the vascular arterial clusters from Crock & Yoshizawa (1976) to high density nerve regions in the center portion of the nerve density graph. (C) An image of a cluster of innervated vessels from a coronal section taken of the center of the vertebral body, verifying the presence of the central arterial clusters within the L4 vertebra.

Fig. 6

L5 Mid-Sagittal Nerve Area is an average of nerve area (mm²) results amongst three mid-sagittal sections from an L5 vertebral body. The sagittal sections demonstrate a similar pattern of nerves, mainly residing in the basivertebral foramen and center of the vertebral body.

Fig. 7

Top image shows the nerve area (mm²) distribution averaged across five mid-coronal sections from the L4 specimen. Bottom image is a graph quantifying the nerve area along the equatorial plane of the same five mid-coronal sections, demonstrating the higher quantity of nerve in the center compared to the periphery.