Yasuchika Aoki

Pain-related sensory innervation in monoiodoacetate-induced osteoarthritis in rat knees that gradually develops neuronal injury in addition to inflammatory pain

BackgroundThe exact mechanism of knee osteoarthritis (OA)-associated pain is unclear, whereas mixed evidence of inflammatory pain and neuropathic pain has been noted. We aimed to investigate pain-related sensory innervation in a monoiodoacetate (MIA)-induced model of OA.MethodsSixty of seventy female Sprague Dawley rats of six week-old underwent intra-articular MIA and fluorogold (FG) retrograde neurotracer injection into their right (ipsilateral) knee, while their left knees were treated with FG in saline as a control (contralateral knee). Other rats were treated with FG only bilaterally, and used as controls. Rats were evaluated for tactile allodynia using von Frey hairs. Proinflammatory mediators in the knee soft tissues, including tumor necrosis factor (TNF)-α, interleukin (IL)-6, and nerve growth factor (NGF), were quantified using ELISAs to evaluate inflammation in the knee after 1, 4, 7,14,21, and 28 days post injection:. Dorsal root ganglia (DRG) were immunostained for three molecules after 7,14,21, and 28 days post injection: calcitonin gene-related peptide (CGRP), a marker of inflammatory pain; and activating transcription factor-3 (ATF3) and growth associated protein-43 (GAP43), as markers for nerve injury and regenerating axons. The distribution of microglia in the spinal cord were also evaluated, because they have been reported to increase in neuropathic pain states. These evaluations were performed up to 28 days postinjection. P < 0.05 was considered significant.ResultsProgressive tactile allodynia and elevated cytokine concentrations were observed in ipsilateral knees. CGRP-immunoreactive (-ir) ipsilateral DRG neurons significantly increased, peaking at 14 days postinjection, while expression of FG-labeled ATF3-ir or ATF3-ir GAP43-ir DRG neurons significantly increased in a time-dependent manner. Significant proliferation of microglia were found with time in the ipsilateral dorsal horn.ConclusionsPain-related characteristics in a MIA-induced rat OA model can originate from an inflammatory pain state induced by the local inflammation initiated by inflammatory cytokines, and that state will be followed by gradual initiation of neuronal injury, which may induce the neuropathic pain state.

Proinflammatory cytokines stimulate the expression of nerve growth factor by human intervertebral disc cells.

Study Design. In vitro studies of the effects of proinflammatory cytokines on the production of nerve growth factor (NGF) by human intervertebral disc (IVD) cells. Objective. To determine the constitutive expression and production of NGF and the effect of cytokines on the expression of NGF by human IVD cells. Summary of the Background Data. NGF may play a role in the collateral sprouting of sensory axons, neural survival, and regulation of nociceptive sensory neurons. NGF is known to be up-regulated by proinflammatory cytokines. Methods. The presence of NGF protein was analyzed by immunohistochemistry using human IVD cells obtained from cadaveric human spines with no known disc disease (MRI Thompson grades 2–4). The effects of interleukin-1&bgr; (IL-1&bgr;) and tumor necrosis factor-&agr; (TNF-&agr;) on NGF production and mRNA expression of NGF by IVD cells were examined. The expression of NGF receptors, trkA and p75NGFR, was also assessed immunohistochemically. Results. Cadaveric anulus fibrosus (AF) and nucleus pulposus (NP) cells cultured in vitro in monolayer and in alginate beads positively stained with an anti-NGF antibody. The constitutive production of NGF protein in IVD cells was low (NP) or not detectable (AF). The expression of NGF mRNA was detectable in both cell types. IL-1&bgr; and TNF-&agr; up-regulated the NGF mRNA expression and the secretion of NGF protein into the media. TrkA was immunolocalized in AF and NP cells. Conclusion. Our results demonstrate, for the first time, that human AF and NP cells constitutively express NGF protein and mRNA, and that the proinflammatory cytokines IL-1&bgr; and TNF-&agr; stimulate the production of NGF. The precise role of NGF produced by IVD cells in the generation of discogenic pain or on the metabolism of IVD cells, especially under certain physiologic conditions in which cytokines are up-regulated, needs to be clarified in future experimentation.

The degenerated lumbar intervertebral disc is innervated primarily by peptide-containing sensory nerve fibers in humans

Study Design. Immunohistochemical study of the sensory innervation of the human lumbar intervertebral disc. Objective. To determine the type of sensory fibers innervating human degenerated lumbar intervertebral discs. Summary of Background Data. Sensory neurons involved in pain perception related to inflammation in rats are typically small, peptide-containing neurons immunoreactive for calcitonin gene-related peptide (CGRP). Small non-peptide-containing neurons binding to isolectin B4 (IB4) may also be involved in pain states, such as nerve injury pain. The character of such sensory neurons in humans has not been clarified. Methods. A degenerated, painful lumbar intervertebral disc was harvested from each of 8 patients during surgery. Sections were immunostained for protein gene product 9.5 (PGP 9.5, a general neuronal marker), CGRP, and IB4. The numbers of PGP 9.5- and CGRP-immunoreactive, and IB4-binding nerve fibers in the discs were counted. Results. PGP 9.5-immunoreactive fibers were observed in all discs. Nerve fibers immunoreactive for CGRP were also observed in 6 of 8 cases. IB4-binding nerve fibers were not found in any case. Conclusions. Almost all of the nociceptive nerve fibers in the human intervertebral disc are peptide-containing nerve fibers, similar to the rat disc, suggesting that nerve fibers related to inflammation may transmit pain originating from human degenerated intervertebral discs.

Innervation of the lumbar intervertebral disc by nerve growth factor-dependent neurons related to inflammatory pain.

Study Design. We used anatomic tracers and immunoreactivity in rats to define dorsal root ganglion neuron populations innervating the lumbar discs in physiologic and inflammatory states. Objectives. To investigate the percentages of calcitonin gene-related peptide-immunoreactive (CGRP-ir) and isolectin B4 (IB4)-binding neurons innervating lumbar discs. Summary of Background Data. Small neurons are classified into two types. One contains CGRP and expresses the nerve growth factor receptor. The other binds IB4 and expresses the glial cell line-derived neurotrophic factor receptor. Methods. A neurotracer, Fluoro-Gold, was applied to the L5–L6 disc in rats. Five days later, 50-μL saline (control group: n = 8) or Complete Freund’s adjuvant (inflammatory group: n = 8) was applied to the disc. Seven days after the second operation, T13–L5 dorsal root ganglions were processed for double staining of CGRP and IB4. Results. Of the Fluoro-Gold-labeled neurons, 50.1 ± 4.6% (mean ± SEM) were positive for CGRP and 0.7 ± 0.6% positive for IB4 in the control group, while 65.6 ± 4.7% were positive for CGRP and 1.0 ± 1.0% positive for IB4 in the inflammatory group. The percentage of CGRP-ir neurons was significantly higher than that of IB4-binding neurons in both groups (P < 0.001, each). The percentage of CGRP-ir neurons in the inflammatory group was significantly higher than in the control group (P < 0.05). Conclusions. We found that most small neurons innervating the disc were CGRP-ir. Furthermore, disc inflammation caused an increase in CGRP-ir neurons but not IB4-binding neurons, suggesting that CGRP-ir, nerve growth factor-dependent neurons are more responsible for discogenic pain.

Epidural administration of spinal nerves with the tumor necrosis factor-alpha inhibitor, etanercept, compared with dexamethasone for treatment of sciatica in patients with lumbar spinal stenosis: a prospective randomized study.

Study Design. Prospective randomized trial. Objective. To examine the effect of the tumor necrosis factor alpha (TNF-&agr;) inhibitor, etanercept, on radicular pain by its epidural administration onto spinal nerves in patients with lumbar spinal stenosis. Summary of Background Data. TNF-&agr; is thought to play a crucial role in the radicular pain caused by lumbar disc herniation and spinal stenosis. Intravenous infusion of infliximab for sciatica has been examined in 2 studies; however, the results were equivocal. Methods. Eighty patients with low back and radicular leg pain were investigated. We diagnosed the patients by physical examination, and X-ray and magnetic resonance imaging. In 40 patients, we epidurally administered 2.0 mL of lidocaine and 10 mg of etanercept onto the affected spinal nerve, and 2.0 mL of lidocaine and 3.3 mg of dexamethasone was used in 40 patients. Low back pain, leg pain, and leg numbness were evaluated using a visual analogue scale (VAS) and Oswestry Disability Index (ODI) score before and for 1 month after epidural administration. Results. Low back pain, leg pain, and leg numbness in the 2 groups were not significantly different before epidural administration. Epidural administration of etanercept was more effective than dexamethasone for leg pain (3 days, and 1, 2, and 4 weeks: P < 0.05), low back pain (3 days, and 1 and 2 weeks: P < 0.05), and leg numbness (3 days, and 1 and 2 weeks: P < 0.05). No adverse event was observed in either group. Conclusion. Our results indicate that epidural administration of a TNF-&agr; inhibitor onto the spinal nerve produced pain relief, but no adverse event. TNF-&agr; inhibitors may be useful tools for the treatment of radicular pain caused by spinal stenosis.

Existence of a Neuropathic Pain Component in Patients with Osteoarthritis of the Knee

Purpose Pain from osteoarthritis (OA) is generally classified as nociceptive (inflammatory). Animal models of knee OA have shown that sensory nerve fibers innervating the knee are significantly damaged with destruction of subchondral bone junction, and induce neuropathic pain (NP). Our objective was to examine NP in the knees of OA patients using painDETECT (an NP questionnaire) and to evaluate the relationship between NP, pain intensity, and stage of OA. Materials and Methods Ninety-two knee OA patients were evaluated in this study. Pain scores using Visual Analogue Scales (VAS), Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC), painDETECT, duration of symptoms, severity of OA using the Kellgren-Lawrence (KL) system, and amount of joint fluid were evaluated and compared using a Spearmans correlation coefficient by rank test. Results Our study identified at least 5.4% of our knee OA patients as likely to have NP and 15.2% as possibly having NP. The painDETECT score was significantly correlated with the VAS and WOMAC pain severity. Compared with the painDETECT score, there was a tendency for positive correlation with the KL grade, and tendency for negative correlation with the existence and amount of joint fluid, but these correlations were not significant. Conclusion PainDETECT scores classified 5.4% of pain from knee OA as NP. NP tended to be seen in patients with less joint fluid and increased KL grade, both of which corresponded to late stages of OA. It is important to consider the existence of NP in the treatment of knee OA pain.

Disc inflammation potentially promotes axonal regeneration of dorsal root ganglion neurons innervating lumbar intervertebral disc in rats.

Study Design. The expression of growth-associated protein 43 (GAP-43), a marker of axonal growth, in the dorsal root ganglion (DRG) neurons innervating the lumbar intervertebral disc was assessed using the retrograde tracing method and immunohistochemistry. Objectives. To study whether disc inflammation affects GAP-43 expression in DRG neurons innervating the disc in rats. Summary and Background Data. Persistent inflammation and nerve ingrowth into the inner layer of degenerated discs can be a cause of discogenic pain. Although the presence of GAP-43-expressing nerve fibers in painful discs has been reported, the expression of GAP-43 in DRG neurons innervating the disc has not been studied. Methods. Seven days after the application of Fluoro-Gold to the L5–L6 disc, 50 &mgr;L of saline (n = 10, control group) or complete Freund’s adjuvant (n = 10, inflammatory group) was applied to the disc in rats. Ten days after the Fluoro-Gold application, T13–L5 DRGs were double-stained with GAP-43 and either calcitonin gene-related peptide or isolectin B4 (IB4). Results. The percentage of Fluoro-Gold-labeled neurons that were positive for GAP-43 was significantly higher in the inflammatory group (44%) than in the control group (24%, P < 0.001). In both groups, the majority of GAP-43-positive neurons were small and positive for calcitonin gene-related peptide but not IB4. Conclusions. The present results suggest that disc inflammation potentially promotes axonal growth of DRG neurons innervating the disc. In light of the strong correlation between the expression of calcitonin gene-related peptide and nerve growth factor receptor, it is most likely that nerve growth factor-sensitive DRG neurons extend their axons following disc inflammation.

Dermatomes and the central organization of dermatomes and body surface regions in the spinal cord dorsal horn in rats

Dermatomes and the associated central projection fields were studied with the application of fluorescent neurotracer, 1,1′‐dioctadecyl‐3,3,3′,3′‐tetramethylindocarbocyanine perchlorate (DiI), to 21 reference points on rat trunk and hindlimb skin. Segmental distribution and rostrocaudal central level of dorsal root ganglion (DRG) neurons innervating reference points were examined and DiI‐induced fluorescent areas were mapped in the horizontal plane through lamina II of the dorsal horn. Segmental levels of DRG neurons innervating reference points were generally identical to the level determined using dye‐extravasation methods. However, innervation of the first digit was situated in the L4 dermatome, not the L3 reported previously using those methods. Generally, afferents from a reference point projected to a single field in the ipsilateral dorsal horn. Reference points on ventral and dorsal median lines of the trunk were represented bilaterally. Afferents from reference points located on the ventral median line of the hindlimb projected to two separate fields: one on the medial margin of spinal cord segments L2–L5 and the other on the medial half of spinal cord segment L5. From the distribution of central projection fields of reference points, central projection fields of dermatomes were revealed as even in shape and located within corresponding spinal cord segments. The arrangement of peripheral and central fields of dermatomes and body surface regions suggests that peripheral and central projection fields of cutaneous afferent fibers are reshaped from the common prototypical pattern that exhibits an orderly and evenly sequenced arrangement. J. Comp. Neurol. 462:29–41, 2003.

Results of surgery for discogenic low back pain: a randomized study using discography versus discoblock for diagnosis.

Study Design. Randomized, controlled study. Objective. To evaluate the diagnosis of discogenic low back pain (LBP) with discography and discoblock. Summary of Background Data. Discogenic LBP is usually diagnosed by magnetic resonance imaging and discography. However, the reliability of discography is controversial. Previously, we reported the usefulness of discoblock with bupivacaine for diagnosis, and discoblock improved the results of anterior interbody fusion surgery. However, that study was not a randomized, controlled study. Therefore, the purpose of the current study was to compare the results of surgery after diagnosis of LBP by discography and discoblock. Methods. Patients (n = 42) with severe LBP showing L4–L5 or L5–S1 disc degeneration on magnetic resonance imaging were evaluated by discography (1.5 mL of contrast medium) or discoblock (intradisc injection of 0.75 mL of 0.5% bupivacaine). We randomized the patients in turn. Anterior discectomy and interbody fusion were performed in patients who responded to the diagnostic procedures. The visual analogue scale score (0, no pain; 100, worst pain), Japanese Orthopedic Association Score (0, worst pain; 3, no pain), Oswestry Disability Index, and patient satisfaction before and 3 years after surgery were recorded and compared between groups. Results. Twelve patients did not show pain provocation by discography or pain relief by discoblock and were excluded. Fifteen patients who showed pain provocation by discography and 15 patients who experienced pain relief with discoblock were evaluated. Rates of improvement in the visual analogue scale score, Japanese Orthopedic Association Score, and Oswestry Disability Index score in the discoblock group were significantly higher than those in the discography group (P < 0.05) from baseline to 3 years after surgery. Three patients were dissatisfied with surgery after discography compared with one patient after discoblock. Conclusion. Pain relief after injection of a small amount of bupivacaine into the painful disc was a useful tool for the diagnosis of discogenic LBP compared with discography.

Exposure of the nucleus pulposus to the outside of the anulus fibrosus induces nerve injury and regeneration of the afferent fibers innervating the lumbar intervertebral discs in rats.

Study Design. Using a retrograde tracing method and immunohistochemistry, we assessed the expression of activating transcription factor 3 (ATF3), a marker of nerve injury, and growth-associated protein 43 (GAP-43), a marker of axonal growth, in dorsal root ganglion (DRG) neurons innervating the lumbar intervertebral discs in rats. Objectives. To investigate ATF3 and GAP-43 expression in DRGs innervating the intervertebral discs after exposure of the nucleus pulposus to the outside of the anulus fibrosus. Summary of Background Data. Degeneration of lumbar intervertebral discs is considered as a cause of low back pain. We speculated that exposure of the nucleus pulposus to the outside of the anulus fibrosus may induce nerve injury and ingrowth into the disc. Methods. A neurotracer, Fluoro-Gold (F-G), was applied to the ventral aspect of L5–L6 intervertebral discs in 20 rats. The rats were classified into 2 groups: an NP group whose disc was punctured to expose the nucleus pulposus (n = 10) and a sham-operated group whose anulus fibrosus surface was scratched superficially (n = 10). Ten days after surgery, bilateral L1–L5 DRGs were processed for staining of ATF3 and GAP-43. Results. In the NP group, 13.9% ± 2.9% of the F-G-labeled neurons innervating the discs were positive for ATF3, while 19.3% ± 2.7% were positive for GAP-43. In contrast, in the sham-operated group, only 0.8% ± 0.4% of the F-G-labeled neurons were positive for ATF3 while 7.4% ± 1.7% were positive for GAP-43. The percentage of both ATF3-immunoreactive (IR) and GAP-43-IR neurons in the NP group was significantly higher than in the sham-operated group (P < 0.05). Conclusions. ATF3-IR and GAP-43-IR neurons were significantly increased in the NP group. These results suggested that exposure of the nucleus pulposus to the outside of the anulus fibrosus induced nerve injury and in growth into the discs. These findings may explain discogenic lower back pain in patients with lumbar disc degeneration.