Upregulation of BDNF and NGF in cervical intervertebral discs exposed to painful whole-body vibration

Abstract

Study design: In vivo study defining expression of the neurotrophins, brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF), in cervical intervertebral discs after painful whole-body vibration (WBV).

Objective: The goal of this study is to determine if BDNF and NGF are expressed in cervical discs after painful WBV in a rat model.

Summary of background data: WBV is a possible source of neck pain and has been implicated as increasing the risk for disc disorders. Typically, aneural regions of painful human lumbar discs exhibit hyperinnervation, suggesting nerve ingrowth as potentially contributing to disc degeneration and pain. BDNF and NGF are upregulated in painfully degenerate lumbar discs and hypothesized to contribute to this pathology.

Methods: Male Holtzman rats underwent 7 days of repeated WBV (15 Hz, 30 min/d) or sham exposures, followed by 7 days of rest. Cervical discs were collected for analysis of BDNF and NGF expression through RT-qPCR and Western blot analysis. Immunohistochemistry also evaluated their regional expression in the disc.

Results: Vibration significantly increases BDNF messenger ribonucleic acid (mRNA) levels (P=0.036), as well as total-NGF mRNA (P=0.035). Protein expression of both BDNF (P=0.006) and the 75-kDa NGF (P=0.045) increase by nearly 4- and 10-fold, respectively. Both BDNF mRNA (R=0.396; P=0.012) and protein (R=0.280; P=0.035) levels are significantly correlated with the degree of behavioral sensitivity (i.e., pain) at day 14. Total-NGF mRNA is also significantly correlated with the extent of behavioral sensitivity (R=0.276; P=0.044). Both neurotrophins are most increased in the inner annulus fibrosus and nucleus pulposus.

Conclusion: The increases in BDNF and NGF in the cervical discs after painful vibration are observed in typically aneural regions of the disc, consistent with reports of its hyperinnervation. Yet, the induction of nerve ingrowth into the disc was not explicitly investigated. Neurotrophin expression also correlates with behavioral sensitivity, suggesting a role for both neurotrophins in the development of disc pain.

Level of evidence: N/A.

Figure 1

Hematoxylin (blue) and eosin (pink) stain schematic of cervical disc in the sagittal plane showing the IAF, OAF, NP and endplate regions of the IVD. Scale bar is 100μm.

Figure 2

Transcript levels of BDNF and NGF are upregulated in the cervical disc following WBV. BDNF and total-NGF remain significantly (*p<0.036) higher in vibrated discs compared to shams.

Figure 3

Expression of BDNF and NGF protein as measured by western blot and normalized to GAPDH levels. Following WBV, BDNF and the 75kDa isoform of NGF are each significantly upregulated (*p<0.045) compared to levels in the sham discs. The inset shows representative bands labeled for both neurotrophins and GAPDH.

Figure 4

Increased BDNF and NGF labeling is evident in discs after WBV compared to sham discs. Both neurotrophins are expressed in rounded cells of the OAF and fibroblast-like cells of the IAF and NP. Scale bars are 200μm in full panel and 100μm in the panels on the right.

Figure 5

Positive labeling for both neurotrophins is evident in all regions of the disc. BDNF labeling in the IAF and NP is greater than in the OAF. NGF expression is greater in the NP than the OAF.

Figure 6

Correlations of BDNF and NGF mRNA and protein levels with percent reduction in paw withdrawal threshold (PWT) at day 14. BDNF (*p=0.012) and total-NGF (*p=0.044) mRNA are significantly correlated with greater decreases in PWT Protein levels for both neurotrophins are weakly correlated with PWT, with only BDNF being significant (*p=0.035).