Substance P–containing nerves within the human vertebral body: an immunohistochemical study of the basivertebral nerve☆☆☆

Introduction 

The innervation of the spinal column is extremely complex and incompletely understood. Most published studies have focused on the innervation of the soft tissues about the spine. Previous reports have identified nerve tissue in the facet joint capsule, the posterior longitudinal ligament (PLL), the anterior longitudinal ligament and the intervertebral disc, specifically, within the annulus fibrosus. Innervation of the structures in the posterior region of the spine is provided by branches of the recurrent sinuvertebral nerve; innervation of the structures in the anterior aspect is often derived from the fine branches of the gray ramus communicans 1, 2, 3, 4, 5, 6, 7, 8.

Although reports on the innervation of the soft tissues in and about the spine have been relatively numerous, there has been, until recently, a paucity of information regarding the innervation of the bony elements. The presence of nerve tissue within the haversian systems and periosteum of long bones of animals and humans has been previously documented 2, 9, 10, 11. Sherman [2], in a 1963 case report, demonstrated nerve within the bone marrow of a vertebral body of an individual with neurofibromatosis, and in another with reticulosarcomatosis. More recently, in 1993, Ahmed et al. [12] documented the presence of immunoreactive neural tissue in rat vertebral bodies. However, it was not until 1998 that nerves were definitively and systematically described within human vertebral bodies. In that study, Antonacci et al. [13] histologically evaluated 69 vertebral bodies from 23 individuals. In this histologic study, intraosseous nerves and neurovascular bundles were routinely identified; they were noted to enter the vertebral body through the midline posterior vascular foramen. Because of the consistent close association of the entering nerve with the basivertebral vessels, the nerves were termed the “basivertebral nerves.”

This initial characterization of the basivertebral nerve was by simple histopathologic techniques. The purpose of this present study was twofold: to determine if the basivertebral nerve could be harvested for histologic analysis by microscopically assisted gross dissection and to determine if the nerves contain the peptide substance P.

Materials and methods 

Eighty-six vertebral bodies from 11 fresh and fresh-frozen human cadavers were obtained through the Department of Anatomy at our institution. These 86 levels included 36 cervical vertebrae from C1 through C7, 14 lumbar vertebrae from L1 through L5 and 36 thoracic vertebrae (T1–T12). The soft tissue and posterior elements were removed to expose the thecal sac. The spinal cord, or cauda equina, in the lumbar and sacral area and dura were then removed en bloc to reveal the PLL. The area about the central vascular foramen was then carefully dissected, first superficial, then deep to the PLL. During this dissection, care was taken to identify the origins of the basivertebral nerve before harvesting. Upon identification of the nerve as it entered the vertebral body, it was carefully removed. Gripping the nerve at its base with small forceps or hemostat and then gently applying traction usually allowed a 0.5 to 0.8 cm length of nerve to be removed from the bone, free of adherent osseous tissue. The nerves harvested were usually between 0.25 mm and 0.5 mm in diameter. The specimens were then placed in buffered formalin, embedded into paraffin blocks and sectioned (3 to 5 microns thick). The sections were then prepared with standard hematoxylin and eosin stains, and evaluated by light microscopy. Those sections that were determined to contain well-preserved neural tissue were then stained for S-100 and substance P.

Sections of formalin-fixed and paraffin-embedded tissue were deparaffinized through xylene and rehydrated through decreasing concentrations of alcohol ending in distilled water. Endogenous peroxidase activity was quenched in 0.3% H202 solution for 10 minutes. After steam heat antigen retrieval for 20 minutes in Antigen Target retrieval solution (DAKO, Carpinteria, CA), blocking of endogenous biotin activity and nonspecific protein binding, the sections were incubated with polyclonal rabbit prediluted, anti–S-100 or anti–substance P antibody by Biogenex (San Ramon, CA) for 30 minutes at room temperature. The bound antibody was detected using Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA) and diaminobenzidine (DAB) as chromogen. Sections were then counterstained in hematoxylin, dehydrated, mounted and coverslipped. Positive control consisted of normal nerve tissue.

Immunohistochemical staining techniques are significantly more demanding in osseous tissue than in soft tissue. Preliminary work in our laboratory had revealed that the decalcification procedures, used in the preparation of histologic section, denatured the substance P in both control and osseous samples. The observation that we could harvest 5- to 10-mm segments of these nerves from within the bone has allowed us to proceed with the initial immunohistochemical studies as described here, without the need for demineralization of the specimen.

Results 

The anatomic dissection revealed that these small nerves entered the vertebral body by means of the basivertebral foramen (Fig. 1). The central vertebral “vascular foramen” was noted to frequently have a vertical bony septum, centrally placed such that two basivertebral nerve and vessel bundles were seen to enter the bone, separated by 2 to 4 mm with an intervening small bony septum. The nerves appear to originate from a small 4 to 6 mm plexus of nerves, associated with a venous plexus located beneath the PLL posterior to the foramen. (Fig. 2).

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Fig. 1. The basivertebral foramen as seen on (Left) a human vertebral anatomic specimen and (Right) a clinical magnetic resonance image. The basivertebral foramen is indicated by arrows.

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Fig. 2. (Top) A plexus of nerves and vessels, anterior to the posterior longitudinal ligament and immediately posterior to the basivertebral foramen. The nerves and vessels are demonstrated by gentle retraction by the probe. (Bottom) The basivertebral foramen before (open arrow) and after (closed arrow) harvest of the intraosseous portion of the nerve.

Basivertebral nerve harvesting was attempted on a total of 86 vertebral bodies. From these, 62 specimens were found to contain well-preserved nerve tissue by histologic examination; 24 of the 86 specimens were excluded from the study because of tissue degeneration and “freezer burns,” which resulted in unacceptable histologic quality in the harvested nerves. None of these excluded specimens were processed for immunohistochemical analysis. The 62 remaining specimens were stained with antibodies to S-100 antigen, and all 62 of the specimens were noted to stain positively for this well-established nerve protein marker using immunoperoxidase technique. This work demonstrated the feasibility of immunohistochemical staining in the basivertebral nerve specimens.

The 62 specimens were then stained for the presence of substance P. All 62 specimens were positive for substance P. The presence of substance P within the intraosseous nerves was universal; no substance P nonreactive nerves were seen (Fig. 3).

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Fig. 3. (Top) Histologic appearance of the basivertebral nerve harvested from within the vertebral body, stained with hematoxylin and eosin. (Bottom) A nerve from this same specimen stained for substance P by immunoperoxidase technique.

Discussion 

Substance P is a peptide neurotransmitter of the tachykinin family. It is released in response to nociceptive stimuli 6, 12, 14, 15. The presence of substance P within the basivertebral nerve is very strong evidence that these nerves have the potential for transmitting pain signals.

The dense innervation of the interior of the vertebral body has been a very recent discovery. The abundance of these nerves and their size suggests that they likely perform multiple functions. Possible functions for these nerves might include regulation of the vasculature and regulation of bone metabolism. Sensation, to include nociception, is another possibility.

The literature offers sparse but undeniable evidence that the vertebral bone itself can be a source of spinal pain. Several authors have noted that during discography procedures, the needle that is introduced into the disc space will provoke intense pain if it impinges the bony end plate 16, 17, 18, 19. Similarly, Kuslich et al. [21] reported on the anatomic sources of pain identified while performing lumbar laminectomies under local anesthesia; mechanical stimulatus of the bony end plate “frequently resulted in a deep, rather severe back pain.”

As the vertebral bone itself clearly can give rise to pain, it is highly probable that these nocioceptive signals are transmitted by means of these basivertebral nerves. Events within the bone, such as microtrabecular fracture, abnormal bony strains, chemical irritation of the bone or abnormal pressure fluctuation, might lead to such pain.

Arnoldi [22], in 1976, proposed that intraosseous hypertension might be a cause of low back pain. Subsequently, both Hanaik et al. [23] and Esses et al. [24] independently described increased intraosseous pressures in degenerated lumbar spine segment in vivo. Further, both groups of investigators reported that increasing vertebral intraosseous pressure by the injection of saline by means of intraosseous catheters resulted in back pain. The thought that local pressure fluctuations may play a role in some clinical back pain patients is made more plausible by the observation that measurable vertebral end plate deflection occurs under physiologic axial loads as well as during discography [25].

It is not known at present whether osseous sources are responsible for any clinically significant back pain. More research on this topic is needed. If it emerges that a subset of patients with back pain has an intraosseous source of pain, a better understanding of the basivertebral nerve may lead to new and better treatment options.20