Gibberellin-biosynthetic ent-kaurene synthases in higher plants do not require their non-catalytic domains for the catalysis.

ent-Kaurene is a biosynthetic intermediate diterpene of phytohormone gibberellins, and is biosynthesized from geranylgeranyl diphosphate via ent-copalyl diphosphate (ent-CDP). The successive cyclization is catalyzed by two distinct diterpene synthases, ent-CDP synthase (ent-CPS) and ent-kaurene synthase (KS). Homologs of these diterpene synthase genes have been reported to be involved in the biosynthesis of specialized-metabolic diterpenoids for defense in several plant species, including rice (Oryza sativa). These diterpene synthases consist of three domains, αßγ domains. Active sites of ent-CPS exist at the interface of ß and γ domain, while those of KS are located within the α domain. We herein carried out domain-deletion experiments using several KSs and KS like enzymes (KSLs) to obtain insights into the roles of domains other than active-site domains. As previously reported in taxadiene synthase, deletion of γ or ßγ domains drastically decreased activities of specialized-metabolic OsKSL5, OsKSL8, OsKSL7 and OsKSL10 in O. sativa. However, unexpectedly, only α domains of several gibberellin-biosynthetic KSs, including OsKS1 in O. sativa, AtKS in Arabidopsis thaliana, TaKS in wheat (Triticum aestivum) and BdKS1 in Brachypodium distachyon, retained their original functions. Additionally, the specialized-metabolic OsKSL4, which is closely related to OsKS1, also functioned without its ßγ domains. Domain-swapping experiments showed that replacing ßγ domains in OsKSL7 with those from other KS/KSLs retained the OsKSL7 activity. Moreover, deletion of ßγ domains of bifunctional PpCPS/KS in moss (Physcomitrella patens) drastically impaired its KS-related activity. Thus, we demonstrate that monofunctional gibberellin-biosynthetic KSs are the unique diterpene synthases that retain their functions without ßγ domains.

Accuracy of cup placement and pelvic motion in total hip arthroplasty in the lateral decubitus position using a new computed tomography-based navigation system with augmented reality technology.

INTRODUCTION: This study aimed to investigate the accuracy of cup position and assess the changes in pelvic tilt during primary total hip arthroplasty (THA) in the lateral decubitus position using a new computed tomography (CT)-based navigation system with augmented reality (AR) technology. MATERIALS AND METHODS: There were 37 cementless THAs performed using a CT-based navigation system with AR technology in the lateral decubitus position and 63 cementless THAs performed using manual implant techniques in the lateral decubitus position in this retrospective study. Postoperative cup radiographic inclination and anteversion were measured using postoperative CT, and the proportion of hips within Lewinnek's safe zone was analyzed and compared between the two groups. The mean absolute values of navigation error were assessed. Intraoperative pelvic tilt angles were also recorded using navigation system. RESULTS: The percentage of cups inside Lewinnek's safe zone was 100% in the navigation group and 35% in the control group (p < 0.001). The mean absolute values of navigation error in inclination and anteversion were 2.9° ± 2.1° and 3.3° ± 2.4°, respectively. The mean abduction angle of the pelvis was 5.1° ± 4.8° after placing the patients in the lateral decubitus position and 4.1° ± 6.0° after cup placement. The mean posterior tilt angle was 6.8° ± 5.1° after placing the patients in the lateral decubitus position and 9.3° ± 5.9° after cup placement. The mean internal rotation angle was 14.8° ± 7.4° after cup placement. There were no correlations between the navigation error in inclination or anteversion and the absolute values of changes of the pelvic tilt angle at any phase. CONCLUSIONS: Although progressive pelvic motion occurred in THA in the lateral decubitus position, especially during cup placement, the CT-based navigation system with AR technology improved cup placement accuracy.

Comparison of effectiveness of methotrexate in patients with late-onset versus younger-onset rheumatoid arthritis: Real-world data from an inception cohort in Japan (NICER-J).

OBJECTIVE: To compare the effectiveness of methotrexate (MTX) as initial therapy in patients with late-onset and younger-onset rheumatoid arthritis (LORA and YORA). METHODS: Of 114 patients with YORA and 96 patients with LORA, defined as RA occurring at ≥65 years of age, enrolled in a multicentre RA inception cohort study, 71 and 66 patients who had been followed up to 6 months after starting MTX treatment were included in this study. RESULTS: Proportions of patients on MTX treatment at 6 months were 96% and 92% in the YORA and LORA groups, respectively. Despite lower doses of MTX in the LORA group compared with the YORA group, no significant difference was observed in clinical disease activity index scores between the two groups throughout the follow-up period. The proportion of patients in clinical disease activity index remission at 6 months was 35% in both groups. Logistic regression analysis revealed that knee joint involvement and high Health Assessment Questionnaire-Disability Index were significant negative predictors of achieving clinical disease activity index remission at 6 months in the LORA group. CONCLUSION: Observations up to 6 months revealed that the effectiveness of MTX administered based on rheumatologist discretion in patients with LORA is comparable to that in patients with YORA in clinical settings.

Long-term results of hemi-resurfacing and metal-on-metal hip resurfacing for osteonecrosis of the femoral head.

PURPOSE: Hemi-resurfacing arthroplasty (Hemi) and metal-on-metal hip resurfacing arthroplasty (HR) were proposed as alternatives to conventional total hip arthroplasty (THA) for patients with osteonecrosis of the femoral head (ONFH). Long-term results were evaluated. METHODS: Twenty-three hips with ONFH were treated, using Hemi in 12 and HR in 11. Mean follow-up was 15 years in the Hemi group and 10 years in the HR group. Long-term outcomes were reviewed retrospectively. RESULTS: In the Hemi group, 8 hips showed acetabular protrusion and were revised to THA. One of the 8 hips showed femoral loosening. In the HR group, pseudotumor was detected in 4 hips (36%), and 1 hip was revised due to symptomatic pseudotumor. No evidence of any femoral or acetabular loosening was seen in the HR group. Ten-year survival rates were 64.2% and 90.9% in the Hemi and HR groups, respectively. Survival rate in the Hemi group dropped to 22.9% at 15 years. CONCLUSIONS: Long-term results of Hemi for patients with ONFH were very poor, and Hemi should not continue to be used. Although HR had a concern of pseudotumor, it could offer attractive indications for 10 years.

Choice of and response to treatment in patients with early-diagnosed rheumatoid arthritis: Real-world data from an inception cohort in Japan (NICER-J).

OBJECTIVE: Various guidelines recommend that patients with early rheumatoid arthritis (RA) try to achieve clinical remission within 6 months, and early therapeutic intervention is important to this end. This study aimed to investigate short-term treatment outcomes of patients with early-diagnosed RA in clinical practice and to examine predictive factors for achieving remission. METHODS: Of the 210 patients enrolled in the multicenter RA inception cohort, 172 patients who were followed up to 6 months after treatment initiation (baseline) were included. Logistic regression analysis was used to examine the impact of baseline characteristics on achievement of Boolean remission at 6 months. RESULTS: Participants (mean age, 62 years) initiated treatment after a mean of 19 days from RA diagnosis. At baseline and 3 and 6 months after treatment initiation, proportions of patients using methotrexate (MTX) were 87.8%, 89.0%, and 88.3%, respectively, and rates of Boolean remission were 1.8%, 27.8%, and 34.5%, respectively. Multivariate analysis revealed that physician global assessment (PhGA) (Odds ratio (OR): 0.84, 95% confidence interval (CI): 0.71-0.99) and glucocorticoid use (OR: 0.26, 95% CI: 0.10-0.65) at baseline were independent factors that predicted Boolean remission at 6 months. CONCLUSION: After a diagnosis of RA, satisfactory therapeutic effects were achieved at 6 months after the initiation of treatment centered on MTX according to the treat to target strategy. PhGA and glucocorticoid use at treatment initiation are useful for predicting the achievement of treatment goals.

Association of bisphosphonate with bone loss and pain-related behavior in an adjuvant-induced osteoporosis model.

OBJECTIVES: Osteoporosis animal models are used extensively to determine the mechanisms of disease pathology and identify potential biological targets. The study aimed to establish a bone loss model, identify pain-related behavior in neighboring joints using an adjuvant-induced osteoporosis (AO) model, and examine the therapeutic effect of bisphosphonates (BP). METHODS: Complete Freund's adjuvant (CFA) was injected subcutaneously into the back of the right foot of 8-week-old female ddY mice. Subsequently, pain, arthritis, and bone density in the right knee were monitored over time. RESULTS: Pain evaluation using von Frey filaments showed a significantly exacerbated knee pain threshold compared to the control group (saline administration) at 7-day and 14-day intervals after CFA administration, and bone density during the same period also significantly declined. The AO model was created similarly; alendronate (ALN) 40 µg/kg was subcutaneously injected twice and vehicle once from 7 to 14 days after onset. In the ALN administration group on the 14th day, significant improvements in bone density, arthritis, and pain threshold around the knee were observed compared to the untreated group. CONCLUSIONS: ALN may contribute to pain improvement through the simultaneous effects of bone mass improvement and suppression of osteoporotic pain.

Functional kaurene-synthase-like diterpene synthases lacking a gamma domain are widely present in Oryza and related species.

Various diterpene synthases have been functionally identified in cultivated rice (Oryza sativa). These are the homologs of ent-copalyl diphosphate (ent-CDP) synthase and ent-kaurene synthase (KS) that are responsible for the biosynthesis of gibberellins, diterpenoid phytohormones. We isolated a cDNA encoding full-length OsKSL12, a previously uncharacterized KS like (KSL) enzyme that consists of a ß-domain and an α-domain with an active center, but lacks an N-terminal γ-domain. Functional analysis using a bacterial expression system showed that recombinant OsKSL12 converted ent-CDP into ent-manool or ent-13-epi-manool. Comparative genomics revealed that functional OsKSL12 homologs exist in diverse wild species in the Oryzeae-Oryza nivara (Oryza rufipogon), Oryza coarctata, Oryza granulata, Leersia perrieri, and Leersia tisseranti. KSL12 homologs in O. granulata, L. perrieri, and L. tisseranti preferentially reacted with geranylgeranyl diphosphate rather than ent-CDP, resulting in geranyllinalool rather than ent-manool or ent-13-epi-manool as the main product, meaning that KSL12 functionally diversified during evolution in the Oryzeae.

Accuracy of acetabular cup insertion in an anterolateral supine approach using an accelerometer-based portable navigation system.

The purpose of this study was to determine the accuracy of acetabular cup insertion during total hip arthroplasty (THA) in a supine position using an accelerometer-based portable navigation system. A single-surgeon study was conducted in which 62 prospective patients with navigation and 42 retrospective patients without navigation as historical controls were compared. The patients underwent THA via an anterolateral supine approach. The absolute values of errors of radiographic inclination and anteversion were calculated. The navigation error was also calculated. The factors that affected the absolute values of errors of cup alignment were determined. The mean absolute error of the postoperative CT measurement from the target angle for radiographic inclination was 3.8° in the navigation group and 6.6° in the control group (P < 0.001). The mean absolute error for anteversion was 3.3° in the navigation group and 5.9° in the control group (P < 0.001). The mean absolute values of navigation error were 3.7° ± 2.8° in inclination and 3.0° ± 2.6° in anteversion. Changes in the pelvic tilt angle among preoperative, intraoperative, and postoperative were the significant risk factors for the absolute values of navigation error of anteversion relative to the anterior pelvic plane. Other factors affecting the absolute values of errors in the navigation group were not found for radiographic inclination and anteversion. The portable navigation system significantly improved the accuracy of cup inclination and anteversion. Changes in the pelvic tilt were the risk factors for the error of anteversion relative to the anterior pelvic plane.

The accuracy of acetabular cup placement in primary total hip arthroplasty using an image-free navigation system.

BACKGROUND: Intraoperative navigation systems have been shown to improve the accuracy of acetabular component insertion in total hip arthroplasty (THA). The purpose of this study was to investigate the accuracy of cup orientation in primary THA using an image-free navigation system. METHODS: A total of 107 consecutive cementless THAs using an image-free navigation system were performed from February 2017 to March 2020 (the navigation group). As a control group, 77 retrospective consecutive cases who underwent THAs with manual implant-techniques between February 2012 and April 2017 were included. Postoperative cup radiographic inclination and radiographic anteversion relative to the functional pelvic plane were assessed using a 3D-template system after computed tomography (CT) examination. RESULTS: The mean absolute errors of the postoperative measured angles from the target angles in inclination were 3.4° ± 3.0° in the navigation group and 8.4° ± 6.6° in the control group (p < 0.001). The mean absolute errors in anteversion were 5.1° ± 3.6° in the navigation group and 10.8° ± 6.5° in the control group (p < 0.001). The percentage of cups inside the Lewinnek safe zone was 93% in the navigation group and 44% in the control group (p < 0.001). The mean absolute values of navigation error were 3.3° ± 2.8° in inclination and 5.8° ± 4.9° in anteversion. Among the cases of osteoarthritis, the inclination error was significantly higher in Crowe group 2 to 4 than in Crowe group 1 (5.1° ± 3.5° and 3.0° ± 2.5°, respectively, p < 0.05). The percentage of hips with inclination error over 10° in Crowe group 2 to 4 was significantly higher than in Crowe group 1 (17 and 1%, respectively, p < 0.05). CONCLUSIONS: The image-free navigation system improved the accuracy of cup orientation. The accuracy of cup position was less in Crowe group 2 to 4 than in Crowe group 1.

Accuracy of image-free navigation in intraoperative leg length change from total hip arthroplasty using evaluations from 2D and 3D measurements.

BACKGROUND: Leg length discrepancy is one of the most common problems after total hip arthroplasty (THA). The aim of this study was to investigate the accuracy of image-free navigation in intraoperative leg length change (LLC) using evaluations from anteroposterior radiographs (2D measurement) and 3D bone models using CT data (3D measurement). METHODS: One hundred THAs with cementless cups and stems were performed using an image-free navigation system in our hospital. We evaluated the accuracy of image-free navigation based on LLC from 2D and 3D measurements. Furthermore, we also investigated error in absolute value and correlations between 2D and 3D measurements in LLC. RESULTS: The accuracy of image-free navigation based on 2D measurement was 94% within 5 mm and 76% within 3 mm. The accuracy of image-free navigation based on 3D measurement was 92% within 5 mm and 81% within 3 mm. The error in absolute value in LLC between 2D and 3D measurements was 1.7 ± 1.4 mm (range, 0 to 6 mm). A strong correlation was observed between 2D and 3D measurements in the LLC. CONCLUSIONS: In the present study, good accuracy of image-free navigation in intraoperative LLC was confirmed for both evaluation methods from 2D and 3D measurements. In addition, the error in absolute value in the LLC between 2D and 3D measurements was very small, and we observed a strong correlation between 2D and 3D measurements. Based on these results, evaluation of LLC from radiographs was considered sufficient if radiographs can be taken accurately.

Minimum 10-Year Follow-Up of Cementless Total Hip Arthroplasty With a 32-mm Cobalt-Chromium Head on Highly Cross-Linked Polyethylene and a Tapered, Fiber Metal Proximally Coated Femoral Stem.

BACKGROUND: The clinical and radiographic results of cementless total hip arthroplasty using a 32-mm cobalt-chromium head on remelted highly cross-linked polyethylene (HXLPE) and a tapered, fiber metal proximally coated femoral stem were evaluated at a mean follow-up of 12 years. METHODS: A total of 57 cementless total hip arthroplasties using remelted HXLPE combined with a 32-mm cobalt-chromium head, and a tapered, fiber metal proximally coated femoral stem were performed from October 2004 to December 2006. Clinical evaluation was performed using the Merle d'Aubigné and Postel scoring system. Standardized anteroposterior and lateral radiographs of the pelvis and femur without weight-bearing were analyzed. Radiographic measurements of two-dimensional femoral head penetration into the polyethylene were performed with a computerized method. The steady-state wear rates were measured based on the radiographs from the first year after surgery to the final follow-up. RESULTS: The mean Merle d'Aubigné and Postel score improved significantly from 10.7 points preoperatively to 15.6 postoperatively (P < .001). No osteolysis was found around the implant. Stem fixation in all cases showed stable bone ingrowth. Third-degree stress shielding was found in 46% of all stems. The total head penetration rate was 0.05 mm/y, and the steady-state wear rate was 0.01 mm/y. The Kaplan-Meier survivorship with the end point of revision was 95% (95% confidence interval, 85%-98%) at 12 years. CONCLUSION: A 32-mm cobalt-chromium head on remelted HXLPE demonstrated low wear properties, and a tapered, fiber metal proximally coated femoral stem showed good results at long-term follow-up.

Minimum ten-year results in revision total hip arthroplasty using titanium fully porous long stem.

PURPOSE: The fully porous long stem could be used for diaphyseal locking and achieve bypass fixation in revision total hip arthroplasty (THA). This study evaluated long-term results for fully porous long stems made of titanium alloy in revision THA. METHODS: Between 2003 and 2010, 45 consecutive femoral revisions were performed using fully porous long stems. Thirty-eight hips had complete clinical and radiographic data for a mean of 13.8 years (range, 10-16.7 years). Femoral bone loss was Paprosky type II in 14 hips, type IIIA in 18 hips, and type IIIB in six hips. Clinical results were analyzed using the Merle d'Aubigné and Postel scoring system. Femoral stress shielding was graded. Radiological loosening of the femoral component was evaluated. Kaplan-Meier survival analysis was performed with revision for any reason as the end-point. RESULTS: Mean Merle d'Aubigné and Postel score improved significantly from 10.7 before revision to 14.5 at latest follow-up (p < 0.001). Third-degree stress shielding was found in seven hips, and fourth degree in 11 hips. Radiological femoral loosening occurred in one hip, which was revised. Another hip was revised for stem fracture. Kaplan-Meier cumulative survival rate was 94.7% at both ten and 15 years. CONCLUSION: Although stress shielding is a concern with fully porous stems, this stem was useful in revision THA and provided satisfactory long-term results in hips with Paprosky types II, IIIA, and IIIB.

Use of antibiotic-impregnated hydroxyapatite for infection following total knee arthroplasty.

OBJECTIVES: Antibiotic-impregnated hydroxyapatite (HA) is an emerging drug delivery system for the treatment of periprosthetic joint infection (PJI) of the knee. The aim of this study was to evaluate the clinical results of this treatment in patients with PJI following total knee arthroplasty (TKA). METHODS: Participants comprised 14 consecutive patients with PJI of the knee who were scheduled to undergo two-stage knee revision. In the first stage, all components were removed with thorough debridement, and antibiotic-impregnated HA was inserted in the bone and joint space. In the second stage, revision TKA was performed after the eradication of infection. The mean period from the removal of all components and treatment with antibiotic-impregnated HA to revision TKA was 3.4 months. Clinical and radiographic evaluations were performed after a mean follow-up of 6.1 years following revision TKA. Predictors of reinfection were evaluated. RESULTS: Eleven patients had no recurrent infection, whereas 3 patients showed recurrent infection. Knee Society knee score and function score, as well as flexion angles, improved significantly postoperatively. No knees showed osteolysis or loosening after revision TKA. The present study identified no predictors of reinfection. CONCLUSION: Antibiotic-impregnated HA could be effective in treating knee PJI.

Thirteen-year results of cementless total hip arthroplasty with zirconia heads on highly cross-linked polyethylene.

OBJECTIVES: Zirconia ceramic was introduced for femoral heads because of its mechanical strength and fracture toughness. Highly cross-linked polyethylene has been shown to achieve wear reduction compared to conventional polyethylene. The purpose of this study was to investigate the long-term results of cementless total hip arthroplasty with zirconia femoral heads on highly cross-linked polyethylene. METHODS: Radiographic measurements of two-dimensional femoral head penetration into the polyethylene were performed with a computerized method (PolyWare) in 93 hips at a mean follow-up of 13 years. The steady-state wear rates were measured based on the radiographs from the first year after surgery to the final follow-up. RESULTS: The mean Merle d'Aubigné and Postel score had improved significantly from 10.9 points preoperatively to 15.3 postoperatively (p < 0.001). Osteolysis was not found around the implant, and no zirconia heads had fractures. The total head penetration rate was 0.04 mm/year, and the steady-state wear rate was 0.02 mm/year. Kaplan-Meier survivorship with the endpoint of revision was 97% (95% confidence interval [CI], 92% to 99%) at 10 years. CONCLUSION: Zirconia on highly cross-linked polyethylene demonstrated extremely low wear properties at long-term follow-up.

Prevention of bone loss and improvement of pain-related behavior in hind limb-unloaded mice by administration of teriparatide and bisphosphonate.

OBJECTIVES: There are few reports on the comparison between teriparatide (PTH) and bisphosphonate (BP) in terms of osteoporosis pain-related behavior and immunohistochemical findings. The aims of this study were to evaluate skeletal pain associated with osteoporosis and to examine the inhibitory effect of PTH and BP on pain and bone loss in hind limb-unloaded (HU) mice. The mechanism of osteoporotic pain in HU mice was evaluated by examining pain-related behavior and immunohistochemical findings. The effects of PTH and alendronate (ALN), a potent osteoclast inhibitor, on these parameters were also assessed. METHODS: Eight-week-old male ddY mice were tail-suspended for 2 weeks and assigned to four groups: hind limb-loaded (HL) mice with only tail suspension treated with vehicle; HU mice with tail suspension treated with vehicle; HU mice treated with PTH; and HU mice treated with ALN. Starting immediately after reloading, vehicle, PTH, or ALN was injected subcutaneously. After a 2-week treatment, mechanical sensitivity was examined using von Frey filaments. Bilateral hind limbs were removed for micro-computed tomography, immunohistochemical analysis, and messenger RNA (mRNA) expression analysis. RESULTS: HU mice with tail suspension developed bone loss and mechanical hyperalgesia in the hind limbs. The HU mice showed an increased osteoclasts and sclerostin-positive cells in the hind limb bone. Furthermore, PTH and ALN both prevented HU-induced bone loss and mechanical hyperalgesia in the osteoporotic animal models. Histological examination of the hind limb bone revealed that, similar to ALN, PTH inhibited the osteoclasts and sclerostin-positive cells. The mRNA levels of TNFα and IL-6 tended to decrease with ALN or PTH treatment compared with those without any treatment. CONCLUSIONS: Treatment with PTH as well as BP prevented bone loss, mechanical hyperalgesia, osteoclast increase, and osteocyte increase. Similar to BP, the inhibitory effect of PTH on osteoclasts might contribute to the improvement of skeletal pain.

CTLA-4Ig Improves Hyperalgesia in a Mouse Model of Osteoporosis.

This study aimed to evaluate skeletal pain associated with osteoporosis and to examine the inhibitory effects of cytotoxic T lymphocyte-associated antigen-4Ig (CTLA-4Ig) administration in ovariectomized (OVX) mice. Eight-week-old female ddY mice were assigned to three groups: sham-operated mice (SHAM) treated with vehicle, OVX mice treated with vehicle (OVX), and OVX mice treated with CTLA-4Ig (CTLA-4Ig). Vehicle or CTLA-4Ig was injected intraperitoneally, starting immediately after surgery. After 4 weeks of treatment, mechanical sensitivity was examined, and the bilateral hind limbs were removed and evaluated by micro-computed tomography, immunohistochemical analyses, and messenger RNA expression analysis. Ovariectomy induced bone loss and mechanical hyperalgesia in the hindlimbs. CTLA-4Ig treatment prevented bone loss in the hindlimbs compared to vehicle administration in the OVX group. Moreover, mechanical hyperalgesia was significantly decreased in the CTLA-4Ig treatment group in comparison to the OVX group. The expression levels of tumor necrosis factor-α (TNF-α) and sclerostin (SOST), as well as the number of osteoclasts, were increased, and the expression level of Wnt-10b was decreased in the OVX group compared with the SHAM group, whereas these parameters were improved in the CTLA-4Ig group compared with the OVX group. The novelty of this research is that CTLA-4Ig administration prevented bone loss and mechanical hyperalgesia induced by ovariectomy in the hindlimbs.

Functional Block of Interleukin-6 Reduces a Bone Pain Marker but Not Bone Loss in Hindlimb-Unloaded Mice.

Interleukin-6 (IL-6) is widely accepted to stimulate osteoclasts. Our aim in this study was to examine whether the inhibitory effect of IL-6 on bone loss and skeletal pain associated with osteoporosis in hindlimb-unloaded (HU) mice in comparison with bisphosphonate. Eight-week-old male ddY mice were tail suspended for 2 weeks. Starting immediately after reload, vehicle (HU group), alendronate (HU-ALN group), or anti-IL-6 receptor antibody (HU-IL-6i group) was injected subcutaneously. After a 2-week treatment, pain-related behavior was examined using von Frey filaments. The bilateral distal femoral and proximal tibial metaphyses were analyzed three-dimensionally with micro-computed tomography. Calcitonin gene-related peptide (CGRP) expressions in dorsal root ganglion (DRG) neurons innervating the hindlimbs were examined using immunohistochemistry. HU mice with tail suspension developed bone loss. The HU mice showed mechanical hyperalgesia in the hindlimbs and increased CGRP immunoreactive neurons in the L3-5 DRG. Treatment with IL-6i and ALN prevented HU-induced mechanical hyperalgesia and upregulation of CGRP expressions in DRG neurons. Furthermore, ALN but not IL-6i prevented HU-induced bone loss. In summary, treatment with IL-6i prevented mechanical hyperalgesia in hindlimbs and suppressed CGRP expressions in DRG neurons of osteoporotic models. The novelty of this research suggests that IL-6 is one of the causes of immobility-induced osteoporotic pain regardless improvement of bone loss.

Interleukin-6 Inhibitor Suppresses Hyperalgesia Without Improvement in Osteoporosis in a Mouse Pain Model of Osteoporosis.

The aim of this study was to evaluate skeletal pain associated with osteoporosis and examine the inhibitory effect of interleukin-6 (IL-6) on pain in ovariectomized (OVX) mice. The mechanism of osteoporotic pain in OVX mice was evaluated by examining pain-related behavior and immunohistochemistry. The effects of IL-6 receptor inhibitor (IL-6i) on these parameters were also assessed. Eight-week-old female ddY mice were ovariectomized and assigned to three groups: OVX mice treated with vehicle (OVX); OVX mice treated with alendronate (OVX-ALN); and OVX mice treated with anti-IL-6 receptor (anti-IL-6R) antibody (OVX-IL6i). Sham-operated mice were treated with vehicle. Immediately after surgery, vehicle, ALN, or anti-IL-6R antibody was injected subcutaneously. After a 4-week treatment, mechanical sensitivity was examined using von Frey filaments. The bilateral distal femoral metaphyses and proximal tibial metaphyses were analyzed three-dimensionally with micro-computed tomography. Calcitonin gene-related peptide (CGRP) expression in L3-L5 dorsal root ganglion (DRG) neurons was examined using immunohistochemistry. Ovariectomy induced bone loss and mechanical hyperalgesia in the hindlimbs with upregulation of CGRP expression in the DRG neurons innervating the hindlimbs. ALN treatment prevented bone loss, but anti-IL-6R antibody treatment had no effect on bone morphometry compared with that of the OVX group. However, mechanical hyperalgesia and CGRP expression were significantly decreased in the OVX-IL6i and OVX-ALN groups compared with those in the OVX group. Although anti-IL-6R antibody treatment had no effect on ovariectomy-induced bone loss, the treatment prevented ovariectomy-induced mechanical hyperalgesia in the hindlimbs and suppressed CGRP expression in DRG neurons. The results suggest that IL-6 is one of the causes of postmenopausal osteoporotic pain, and anti-IL-6R antibody might preserve bone health and decrease osteoporotic pain.

Decreased sensory nerve excitation and bone pain associated with mouse Lewis lung cancer in TRPV1-deficient mice.

Bone pain is one of the most common and life-limiting complications of cancer metastasis to bone. Although the mechanism of bone pain still remains poorly understood, bone pain is evoked as a consequence of sensitization and excitation of sensory nerves (SNs) innervating bone by noxious stimuli produced in the microenvironment of bone metastases. We showed that bone is innervated by calcitonin gene-related protein (CGRP)+ SNs extending from dorsal root ganglia (DRG), the cell body of SNs, in mice. Mice intratibially injected with Lewis lung cancer (LLC) cells showed progressive bone pain evaluated by mechanical allodynia and flinching with increased CGRP+ SNs in bone and augmented SN excitation in DRG as indicated by elevated numbers of pERK- and pCREB-immunoreactive neurons. Immunohistochemical examination of LLC-injected bone revealed that the tumor microenvironment is acidic. Bafilomycin A1, a selective inhibitor of H+ secretion from vacuolar proton pump, significantly alleviated bone pain, indicating that the acidic microenvironment contributes to bone pain. We then determined whether the transient receptor potential vanilloid 1 (TRPV1), a major acid-sensing nociceptor predominantly expressed on SNs, plays a role in bone pain by intratibially injecting LLC cells in TRPV1-deficient mice. Bone pain and SN excitation in the DRG and spinal dorsal horn were significantly decreased in TRPV1 -/- mice compared with wild-type mice. Our results suggest that TRPV1 activation on SNs innervating bone by the acidic cancer microenvironment in bone contributes to SN activation and bone pain. Targeting acid-activated TRPV1 is a potential therapeutic approach to cancer-induced bone pain.

The effects of bisphosphonate on pain-related behavior and immunohistochemical analyses in hindlimb-unloaded mice.

OBJECTIVE: The aim of this study was to evaluate skeletal pain associated with immobility-induced osteoporosis and to examine the inhibitory effect of bisphosphonate (BP) administration on pain in hindlimb-unloaded (HU) mice. METHODS: The mechanism of osteoporotic pain in HU mice was evaluated through an examination of pain-related behavior, as well as immunohistochemical findings. In addition, the effects of alendronate (ALN), a potent osteoclast inhibitor, on these parameters were assessed. RESULTS: HU mice with tail suspension developed bone loss and mechanical hyperalgesia in the hindlimbs. The HU mice showed an increase in the number of calcitonin gene-related peptide (CGRP)-immunoreactive neurons and in transient receptor potential channel vanilloid subfamily member 1 (TRPV1)-immunoreactive neurons in the dorsal root ganglions (DRGs) innervating the hindlimbs. Furthermore, administration of ALN prevented HU-induced bone loss, mechanical hyperalgesia, and upregulation of CGRP and TRPV1 expressions in DRG neurons of immobility-induced osteoporotic animal models. CONCLUSIONS: HU mice appear to be a useful model for immobility-induced osteoporotic pain and hindlimb-unloading-induced bone loss, as well as upregulation of CGRP and TRPV1 expressions in DRG neurons, and BP treatment prevented bone loss and mechanical hyperalgesia. The inhibitory effect of BP on osteoclast function might contribute to improving osteoporosis-related pain.