Nerve growth factor regulation and production by macrophages in osteoarthritic synovium

Nerve growth factor (NGF) functions to modulate osteoarthritis (OA)‐associated pain. Although recent studies suggest that tumour necrosis factor (TNF)‐α and interleukin (IL)‐1β mediate NGF activity in human synovial fibroblasts, the regulation of NGF expression in human synovial macrophages remains unclear. Here, we examined the role of macrophages in the production and regulation of synovial (SYN) NGF in osteoarthritic knee joints by examining the mRNA expression of TNF‐α and IL‐1β in freshly isolated CD14‐positive (macrophage‐rich fraction) and CD14‐negative cells (fibroblast‐rich fraction) in synovial tissue from OA patients by quantitative polymerase chain reaction. We also examined the effects of IL‐1β and TNF‐α on NGF mRNA expression in cultured CD14‐positive (macrophage‐rich fraction) and CD14‐negative cells (fibroblast‐rich fraction). In addition, to examine the contribution of macrophages to NGF, TNF‐α and IL‐1β expression, we injected clodronate liposomes systemically into STR/Ort mice, an osteoarthritis animal model, to deplete macrophages. TNF‐α and IL‐1β mRNA levels in CD14‐positive cells from the SYN of OA patients was significantly higher than that in CD14‐negative cells, while NGF expression did not differ markedly between the two cell fractions. In addition, treatment of human cultured CD14‐positive and ‐negative cells with IL‐1β and TNF‐α enhanced NGF mRNA and protein levels. Expression of NGF, IL‐1β and TNF‐α was also reduced significantly in STR/Ort mice upon macrophage depletion. These findings suggest that IL‐1β and TNF‐α regulate NGF expression and production in synovial macrophages and fibroblasts in osteoarthritic joints.     

Efficacy of Combination of Meloxicam and Pregabalin for Pain in Knee Osteoarthritis

Purpose

Osteoarthritic pain is largely considered to be inflammatory pain. Sensory nerve fibers innervating the knee have been shown to be significantly damaged in rat models of knee osteoarthritis (OA) in which the subchondral bone junction is destroyed, and this induces neuropathic pain (NP). Pregabalin was developed as a pain killer for NP; however, there are no reports on pregabalin use in OA patients. The purpose of this study was to investigate the efficacy of pregabalin for pain in OA patients.

Materials and Methods

Eighty-nine knee OA patients were evaluated in this randomized prospective study. Patients were divided into meloxicam, pregabalin, and meloxicam+pregabalin groups. Pain scores were evaluated before and 4 weeks after drug application using a visual analogue scale (VAS), and Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC). Pain scales among groups were compared using a Kruskal-Wallis test.

Results

Before drug application, there was no significant difference in VAS and WOMAC scores among the three groups (p>0.05). Significant pain relief was seen in the meloxicam+pregabalin group in VAS at 1, 2, and 4 weeks, and WOMAC score at 4 weeks, compared with the other groups (p<0.05). No significant pain relief was seen in the meloxicam only group in VAS during 4 weeks and WOMAC score at 4 weeks compared with the pregabalin only group (p>0.05).

Conclusion

Meloxicam+pregabalin was effective for pain in OA patients. This finding suggests that OA pain is a combination of inflammatory and NP.     

Sequential CCL2 Expression Profile After Disc Injury in Mice

Macrophages produce proinflammatory cytokines in injured intervertebral discs (IVDs). We recently showed that macrophage-derived inflammatory cytokines contribute to the production of pain-related factors. However, the mechanism by which macrophages are recruited to injured IVDs has not been fully clarified. Here, we examined the expression dynamics of the chemokine CCL2 in a mouse IVD injury model and the mechanisms of its regulation. The percentage of macrophages increased from day 1 after injury and persisted up until day 28. At 1 and 3 days after injury, the expression of both Ccl2 messenger RNA (mRNA) and CCL2 protein was elevated in the IVD injury group, after which expression decreased to basal levels. Consistent with the increase in CCL2 expression, Ccr2 and Tnfa expression and various types of macrophages were also immediately elevated following disc injury. Further, tumor necrosis factor-α (TNF-α) stimulated Ccl2 mRNA and CCL2 protein expression in IVD cells in vitro. The expressions of M1 (Cd86 and Nos2) and M2a (Ym1) macrophage markers were all significantly elevated from day 1 following injury in injured compared with control mice. Meanwhile, the expression of Cd206 (M2a and M2c marker) was significantly elevated on days 3, 7, 14, and 28 following injury. These results suggest that in IVD injury, TNF-α stimulates CCL2, which, in turn, mediates the recruitment of macrophages with the recruited macrophages subsequently differentiating into M1 and M2 subtypes. CCL2 signaling may, therefore, play an important role in IVD pathology via macrophage recruitment. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:895-901, 2020     

Low affinity NGF receptor (p75 neurotrophin receptor) inhibitory antibody reduces pain behavior and CGRP expression in DRG in the mouse sciatic nerve crush model

Nerve growth factor (NGF) and its low‐affinity receptor, p75 neurotrophin receptor (p75NTR), are important mediators of pain. To explore further the mechanisms involved, we examined suppression of pain behavior and expression of neuropeptides such as calcitonin gene‐related peptide (CGRP) using a p75 NTR inhibitory antibody, in a mouse sciatic nerve crush model. In the nerve‐injured model, 150 µg of a p75 NTR inhibitory antibody or 10 µl of saline were applied. The sciatic nerve in the sham‐operated group was uninjured. Mechanical allodynia was measured for 2 weeks. CGRP and p75NTR expression in L5 dorsal root ganglions (DRGs) was examined immunohistochemically. Mechanical allodynia was found in the two nerve injured groups, but not in the sham‐operated group (p < 0.05). However, the magnitude of the mechanical allodynia was significantly decreased after application of p75 NTR inhibitory antibody (p < 0.05). CGRP and p75NTR immunoreactivity in the L5 DRG neurons was upregulated in the injured nerve groups compared with the sham‐operated group; however, p75 NTR inhibitory antibody decreased the CGRP and p75NTR expression (p < 0.01). Application of the p75 NTR inhibitory antibody to the pinched sciatic nerve suppressed CGRP and p75NTR expression and pain behavior. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:279–283, 2010     

Effectiveness of L2 spinal nerve infiltration for selective discogenic low back pain patients

Background

It has been reported that rat L5/6 lumbar discs are innervated mainly by L2 dorsal root ganglion neurons. We previously reported that L2 spinal nerve infiltration was effective for discogenic low back pain (DLBP) patients, although the diagnosis was based only on the results of physical examination, plain films, and magnetic resonance imaging (MRI). The purpose of the current study was to evaluate L2 spinal nerve block for DLBP patients retrospectively based on MRI findings and surgical results.

Methods

A total of 62 patients with only LBP and no accompanying radicular pain were investigated. Patients had only one level of disc degeneration on MRI. When pain was provoked during discography, we performed surgery at the next stage (40 patients). In all, 22 patients were excluded owing to negative discography results. Of the 40 patients, we evaluated 25 strictly selected patients suffering from DLBP. DLBP was diagnosed when the patient experienced pain relief at least 2 years after anterior lumbar interbody fusion. Fifteen patients who did not show pain relief after surgery were used for the non-DLBP group. L2 spinal nerve infiltration using 1.5 ml of lidocaine was performed in all 40 patients before surgery. The visual analogue scale (VAS) score after L2 spinal nerve infiltration was recorded, and an association of L2 spinal nerve infiltration and DLBP was explored.

Results

Low back pain scores assessed using the VAS score, the Japanese Orthopedic Association score, and the Oswestry Disability Index score in the two groups were not significantly different. L 2 spinal nerve infiltration was effective for 27 patients but not effective for 13 patients; the VAS score after 15 min and 2 h improved in the DLBP group compared with that of the non-DLBP group (P < 0. 05). L2 spinal nerve infiltration was more effective in DLBP patients (21 patients, 84%) than in the non-DLBP group (6 patients, 40%) (P < 0.05).

Conclusions

In the current study, L2 spinal nerve infiltration was effective in 84% of selected DLBP patients and is thought to be a useful tool for diagnosing DLBP. However, we should take into consideration that the L2 spinal nerve infiltration was effective in 40% of non-DLBP patients as well.     

Risedronate decreases bone resorption and improves low back pain in postmenopausal osteoporosis patients without vertebral fractures

Elderly postmenopausal women who have osteoporosis sometimes experience low back pain, however, the relationship between low back pain and osteoporosis in the absence of vertebral fractures remains unclear. We examined the relationship between bone mineral density (BMD), bone resorption and low back pain in elderly female patients who did not have osteoporotic vertebral fractures. The average BMD was 0.675 g/cm² when assessed by dual-energy X-ray absorptiometry (DEXA). Patients were excluded from the study if they had vertebral fractures revealed by radiography, CT scans or MRI. Bisphosphonate (risedronate) was administered for 4 months. The visual analogue scale (VAS) pain score, Roland Morris Disability Questionnaire (RDQ), Short Form-36 (SF-36) questionnaire, BMD and N-terminal telopeptide of type I collagen (NTx; a marker for bone resorption) were examined before and after treatment. DEXA did not increase significantly, but serum and urinary NTx were decreased (?51.4% and ?62.0%, respectively) after 4 months of risedronate treatment (p < 0.01). The assessment was repeated using the VAS score, RDQ and SF-36, which revealed an improvement after risedronate treatment (p < 0.01). A decrease in serum and urinary NTx was associated with improvement of low back pain, suggesting that despite the absence of vertebral fractures, bone resorption due to osteoporosis may cause low back pain.     

Changes in Nerve Growth Factor Expression and Macrophage Phenotype Following Intervertebral Disc Injury in Mice

Nerve growth factor (NGF) is increased in intervertebral discs (IVDs) after disc injury and anti‐NGF therapy improves low back pain in humans. Furthermore, M1 and M2 macrophage subtypes play a role in degenerative IVD injury. We examined M1 and M2 macrophage markers and NGF and cytokine expression in IVD‐derived cells from control and IVD‐injured mice for 28 days following injury. Ngf messenger RNA (mRNA) expression was increased 1 day after injury in injured compared with control mice, and persisted for up to 28 days. Flow cytometric analysis demonstrated that the proportion of F4/80+ CD11b+ cells was significantly increased from 1 day after injury for up to 28 days in injured compared to control mice. mRNA expression of M1 macrophage markers Tnfa, Il1b, and Nos2 was significantly increased 1 day after injury in injured compared to control mice, before gradually decreasing. At 28 days, no significant difference was observed in M1 markers. The M2a marker, Ym1, was significantly increased 1 day after injury in injured compared with control mice, while M2a and M2c markers Tgfb and Cd206 were significantly increased 7, 14, and 28 days after injury. Tumor necrosis factor α (TNF‐α) and transforming growth factor β (TGF‐β) stimulated Ngf mRNA and NGF protein expression in IVD cells. Our results suggest that TNF‐α and TGF‐β may stimulate NGF production under inflammatory and non‐inflammatory conditions following IVD injury. As TNF‐α and TGF‐β are produced by M1 and M2 macrophages, further investigations are needed to reveal the role of macrophages in NGF expression following IVD injury. Our results may aid in developing treatments for IVD‐related LBP pathology. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1798–1804, 2019     

IL-1β mediates NGF and COX-2 expression through transforming growth factor-activating kinase 1 in subacromial bursa cells derived from rotator cuff tear patients

BackgroundIncreased interleukin (IL)-1β expression in the subacromial bursa (SAB) is associated with severe pain in rotator cuff tears (RCTs). Additionally, transforming growth factor (TGF)-β-activated kinase 1 (TAK1) is essential for cytokine-mediated cascades. TAK1 also regulates the expression of pain-associated molecules such as cycloxygenase-2 (COX-2) and nerve growth factor (NGF) in synovial fibroblasts; however, this regulation in the SAB is not fully understood.MethodsSAB samples were harvested from 18 subjects with RCTs. The expression and localization of NGF and COX-2 was determined using polymerase chain reaction (PCR) analysis and immunohistochemistry. Regulation of COX-2 and NGF by IL-1β in subacromial bursa cells (SABCs) was investigated by culturing and stimulating SABCs with vehicle control (culture medium), 50 ng/ml recombinant human IL-1β (rhIL1-β), 50 ng/ml rhIL-1β and 10 μM celecoxib (COX-2 inhibitor), or 10 μM prostaglandin E2 (PGE2) for 24 h. The effects of TAK1 inhibition on rhIL-1β stimulation were determined by culturing and treating SABCs with control, 50 ng/ml rhIL-1β, or 50 ng/ml rhIL-1β and 10 μM (5Z)-7-oxozeaenol (TAK1 inhibitor) for 24 h. NGF and COX-2 mRNA expression was monitored using quantitative PCR.ResultsCOX-2 and NGF mRNA expression was observed in all SAB specimens. Immunohistochemical analysis showed that COX-2-positive cells were in the lining and sublining layers. NGF-positive cells were observed in the sublining layer. rhIL-1β treatment significantly increased NGF and COX-2 mRNA levels compared with control cells. The COX-2 inhibitor did not suppress rhIL-1β-induced NGF expression, and PGE2 stimulation did not alter NGF mRNA expression. In contrast, the TAK1 inhibitor significantly reduced rhIL-1β-stimulated COX-2 and NGF mRNA expression.ConclusionIL-1β regulates the expression of NGF and COX-2, pain-related molecules in the SAB, through TAK1. Therefore, TAK1 may be one potential therapeutic target for reducing pain in patients with RCTs.     

Eosinophilic meningitis: a suspected case of angiostrongylosis found in Shizuoka Prefecture, Honshu, Japan.

A patient with eosinophilic meningitis in Shizuoka Prefecture, Honshu, Japan had nausea, vomiting and headache on admission; laboratory examinations revealed leukocytosis with eosinophilia. Eight days later neck stiffness appeared. Lumbar puncture showed an increase of the initial pressure in association with eosinophilic pleocytosis in the spinal fluid. The possibility of angiostrongylosis was considered because the patient had eaten raw slugs for 4 years as a remedy for lumbago. Although the serum contained cross-reactive antibodies against Toxocara canis, positive reactions to Angiostrongylus cantonensis antigens were observed in all of the immunological tests made. These observations, together with results of epidemiological studies in Honshu carried out by other investigators, suggest that the present case of eosinophilic meningitis may have been caused by A. cantonensis. One previous case has been reported from Honshu Island, Japan.