Mitsuru Hanada

Blockade of IL-6 signaling by MR16-1 inhibits reduction of docosahexaenoic acid-containing phosphatidylcholine levels in a mouse model of spinal cord injury

The interleukin (IL)-6 pathway plays an important role in recovery after spinal cord injury (SCI). The anti-IL-6 receptor antibody MR16-1 has been shown to suppress inflammation after SCI and promote recovery of motor function. The purpose of this study was to analyze the effects of MR16-1 on the expression patterns of phospholipids in the spinal cord in a mouse model of SCI. Eight-week-old C57BL/6JJmsSlc mice were used in this study. Laminectomy was performed at the ninth and tenth thoracic levels (T9-T10), and contusion injury of the spinal cord was induced at level T10. Immediately after SCI, mice were intraperitoneally injected with a single dose of MR16-1 (MR16-1 group) or a single dose of phosphate-buffered saline of the same volume (control group). Imaging mass spectrometry was performed to visualize phosphatidylcholine (PC) expression in the spinal cord 7 days after SCI. We found that MR16-1 treatment suppressed the infiltration of immune cells after SCI, and was able to increase the locomotor function post-injury. Phospholipid imaging revealed that the MR16-1 was able to prevent the reduction of docosahexaenoic acid (DHA)-containing PC in comparison with the control group. We also observed high levels of glial fibrillary acidic protein (GFAP) at the site of DHA-containing PC expression in the MR16-1 group. These results suggest that MR16-1 treatment influences the DHA-containing PC composition of GFAP-positive cells at the injury site as early as 7 days post-SCI.

Increased arachidonic acid-containing phosphatidylcholine is associated with reactive microglia and astrocytes in the spinal cord after peripheral nerve injury.

Peripheral nerve injury (PNI) triggers cellular and molecular changes in the spinal cord. However, little is known about how the polyunsaturated fatty acid-containing phosphatidylcholines (PUFA-PCs) are regulated in the spinal cord after PNI and the association of PUFA-PCs with the non-neuronal cells within in the central nervous system (CNS). In this study, we found that arachidonic acid-containing phosphatidylcholine (AA-PC), [PC(16:0/20:4)+K]+, was significantly increased in the ipsilateral ventral and dorsal horns of the spinal cord after sciatic nerve transection, and the increased expression of [PC(16:0/20:4)+K]+ spatiotemporally resembled the increase of reactive microglia and the astrocytes. From the lipidomics point of view, we conclude that [PC(16:0/20:4)+K]+ could be the main phospholipid in the spinal cord influenced by PNI, and the regulation of specific phospholipid molecule in the CNS after PNI is associated with the reactive microglia and astrocytes.

Reductions of docosahexaenoic acid-containing phosphatidylcholine levels in the anterior horn of an ALS mouse model

In this study, we analyzed the spatiotemporal alterations of phospholipid composition in the spinal cord of an amyotrophic lateral sclerosis (ALS) mouse model (G93A-mutated human superoxide dismutase 1 transgenic mice [SOD1(G93A) mice]) using imaging mass spectrometry (IMS), a powerful method to visualize spatial distributions of various types of molecules in situ. Using this technique, we deciphered the phospholipid distribution in the pre-symptomatic stage, early stage after disease onset, and terminal stages of disease in female SOD1(G93A) mouse spinal cords. These experiments revealed a significant decrease in levels of docosahexaenoic acid (DHA)-containing phosphatidylcholines (PCs), such as PC (diacyl-16:0/22:6), PC (diacyl-18:0/22:6), and PC (diacyl-18:1/22:6) in the L5 anterior horns of terminal stage (22-week-old) SOD1(G93A) mice. The reduction in PC (diacyl-16:0/22:6) level could be reflecting the loss of motor neurons themselves in the anterior horn of the spinal cord in ALS model mice. In contrast, other PCs, such as PC (diacyl-16:0/16:0), were observed specifically in the L5 dorsal horn gray matter, and their levels did not vary between ALS model mice and controls. Thus, our study showed a significant decrease in DHA-containing PCs, but not other PCs, in the terminal stage of ALS in model mice, which is likely to be a reflection of neuronal loss in the anterior horns of the spinal cords. Given its enrichment in dorsal sensory regions, the preservation of PC (diacyl-16:0/16:0) may be the result of spinal sensory neurons being unaffected in ALS. Taken together, these findings suggest that ALS spinal cords show significant alterations in PC metabolism only at the terminal stage of the disease, and that these changes are confined to specific anatomical regions and cell types.

A biochemical study of the distribution of collagen and its crosslinks in knee ligaments and the patellar tendon.

Abstract Purpose: The purpose of this study was to investigate biochemical differences in collagen crosslinks from different locations within the ligaments and a tendon of the human knee. Materials and Methods: The anterior cruciate ligament (ACL), posterior cruciate ligament (PCL), medial collateral ligament (MCL), lateral collateral ligament (LCL), and patellar tendon (PT) were obtained from 24 cadavers (13 men and 11 women) whose average age at the time of death was 84.8 years. Ligaments and PT samples were obtained from the femoral and tibial insertions and the midsubstance. Hydroxyproline (Hyp) and collagen crosslinks, including pyridinoline (Pyr) and pentosidine (Pen), were compared among the different sites. Results: The midsubstance Hyp concentration was greater than at the femoral and tibial insertions in the ACL (p = 0.00124 and 0.000255, respectively) and PCL (p = 0.00036 and 0.042, respectively). The Pyr:collagen ratio did not differ among sites in any of the ligaments or PT. The Pen:collagen ratio at the midsubstance was greater than at the femoral and tibial insertions in the ACL (p = 0.00022 and 0.00025, respectively) and LCL (p = 0.000081 and 0.000021, respectively) and was greater at the femoral insertion in the MCL (p = 0.00010). Conclusions: The mature collagen crosslink Pyr was not different in distribution in knee ligaments and the PT. Pen increased at the midsubstance ligaments and the PT. As increased Pen may represent ligament degeneration, this may indicate that degeneration may progress more rapidly at the midsubstance than at the insertion sites of a ligament.

Relationship between the clinical findings and radiographic severity in Osgood–Schlatter disease

Background Osgood–Schlatter disease (OSD) is one of the common causes of knee pain in active adolescents who play sports. The common age for boys to have OSD is between 12 and 15 years and for girls, between 8 and 12 years. Radiographic studies are helpful in diagnosis and treatment of OSD. Purpose We examine the age at onset of OSD in detail and investigate the relationship between clinical findings, radiographic bone morphology, and the severity of OSD in adolescents. Results The average age at onset of knee pain was 12 years and 6 months – 12 years and 9 months in boys, and 12 years and 1 month in girls. Boys were significantly older than girls at onset. In addition, there were significant relationships between duration from first onset to visit to the clinic, radiographic bone stage, body morphology, and radiographic severity. The patients who delayed their visit to the clinic from the first onset of pain and who were older showed a later bone stage and more radiographic severity grade of OSD. There was significant differences concerning weight and body mass index between severity grade I and III. Conclusion For the age at the onset of OSD, the mean age of boys was significantly older than that of girls. The patients at a later bony stage had a higher severity grade. The boys and girls with OSD who had less weight or body mass index showed less severity.

Evaluation of the effect of tranilast on rats with spinal cord injury

BACKGROUND Glial and fibrotic scars inhibit neural regeneration after spinal cord injury (SCI). N-[3,4-dimethoxycinnamoyl]-anthranilic acid (tranilast) inhibits transforming growth factor β, alleviates allergic reactions, and decreases hypertrophic skin scars. We evaluated its ability to improve motor function and inhibit the spread of tissue damage in rats with SCI. METHODS Rats with SCI were divided into groups that received tranilast (30 mg/[kg · day]) by intravenous administration (group IV), tranilast (200mg/[kg · day]) by oral administration (group OR), and saline injections (control). Motor functions were assessed by determining Basso, Beattie, and Bresnahan (BBB) scores and %grip tests for 8 weeks after SCI. Histological evaluation of ionized calcium binding adaptor molecule 1 (Iba1) at 1 week after SCI and glial fibrillary acidic protein (GFAP), fibronectin, and chondroitin sulfate (CS) at week 8 was performed. RESULTS Motor function recovery, BBB score, and the %grip test were significantly higher in the tranilast-treated groups than in the control group. At week 1 after SCI, inflammatory-cell invasion was more severe and Iba1 expression was significantly higher in the control group. At week 8, although the number of GFAP-positive cells increased greatly from the impaction site to the proximal and distal sites in the control group, these cells were confined around a cavity in the tranilast-treated groups. GFAP distribution coincided with that of fibronectin. Anti-CS antibody level in the tranilast-treated groups was significantly lower than that in the control group. CONCLUSIONS Tranilast inhibits inflammation in the acute phase of SCI and reduces glial and fibrotic scars and could present a new method for treating SCI.

Arundic acid (ONO-2506) inhibits secondary injury and improves motor function in rats with spinal cord injury

BACKGROUND Arundic acid (ONO-2506) inhibits the production and release of S100 protein from astrocytes. While numerous studies have assessed the effect of ONO-2506 in the diseased brain, to the best of our knowledge, no study has examined the effect of ONO-2506 in spinal cord injury (SCI). In this study, we administered ONO-2506 to rats with SCI in order to evaluate its effectiveness in improving motor function and protecting against histological injury. METHODS All rats underwent laminectomy with SCI at the 10th thoracic vertebra. Rats were divided into 3 groups that received different concentrations of ONO-2506 as follows: 10 mg/kg (Group I) and 20 mg/kg (Group II). The third group (control group) was administered only saline. ONO-2506 or saline was administered by intravenous injection for a week after SCI. Recovery of motor function was assessed by determining the Basso, Beattie, and Bresnahan (BBB) scores and using the %grip test. Using immunohistochemistry, S100 protein and glial fibrillary acidic protein expression was assessed at week 12 post SCI. RESULTS The BBB score of Group II was significantly better than that of the control group. At week 12 post SCI, the %grip was 43.0% in Group II and 20.3% in Group I. The score for the %grip test was greater for Group II than for the control group (7.0%); thus, motor function improvement appeared to be dose dependent. Regarding immunostaining evaluation, S100 protein staining was lower in Group II compared to the control group, and the astrocytic morphology resembled that of normal spinal cord sections. The SCI lesion expanded from the injured site to both proximal and distal sites in the control group and in Group I. However, despite the presence of cavitation, secondary expansion of the SCI lesion was prevented in Group II as a result of inhibition of S100 protein. CONCLUSIONS Administration of ONO-2506 (20 mg/kg) improves motor function and inhibits expansion of secondary injury in SCI rats.

Open re-rupture of the Achilles tendon after surgical treatment.

The rate of re-rupture of Achilles tendon after surgical treatment were reported to 1.7–5.6% previously. Re-rupture of Achilles tendon generally occurs subcutaneously. We experienced two rare cases of the open re-ruptures of Achilles tendon with a transverse wound perpendicular to the primary surgical incision. Re-rupture occurred 4 and 13 weeks after surgical treatment. We suggest that open re-rupture correlates more closely with skin scaring and shortening. Another factor may be adhesion between the subcutaneous scar and the suture of the paratenon and Achilles tendon with post-operative immobilization.

Elevated erythrocyte sedimentation rate and high-sensitivity C-reactive protein in osteoarthritis of the knee: relationship with clinical findings and radiographic severity

Purpose We assessed erythrocyte sedimentation rate and high-sensitivity C-reactive protein concentration in knee osteoarthritis and non-knee osteoarthritis. In addition, we investigated potential relationship between the levels of erythrocyte sedimentation rate and high-sensitivity C-reactive protein with clinical findings and radiographic severity. Methods We compared erythrocyte sedimentation rate and high-sensitivity C-reactive protein concentration between 104 patients with knee osteoarthritis (knee osteoarthritis group; 25 males, 79 females; mean age, 73 y) and 50 patients without knee osteoarthritis (non-knee osteoarthritis group; 16 males, 34 females; mean age, 64 y) excluding any patients with comorbid joint osteoarthritis, rheumatoid arthritis, malignant tumours or inflammatory diseases. In the knee osteoarthritis group, we assessed whether erythrocyte sedimentation rate and high-sensitivity C-reactive protein concentration differed in clinical features and Kellgren-Lawrence (KL) grades. Results Erythrocyte sedimentation rate and high-sensitivity C-reactive protein were significantly higher in the knee osteoarthritis group than in the non-knee osteoarthritis group (P = 0.0013 and 0.00010, respectively). In the knee osteoarthritis group, erythrocyte sedimentation rate was significantly elevated in patients with tenderness and patellar ballottement (P = 0.032 and 0.038, respectively), and high-sensitivity C-reactive protein concentration was significantly elevated in patients with tenderness, swelling and patellar ballottement (P = 0.0042, 0.00030 and 0.019, respectively). Erythrocyte sedimentation rate in KL-I was lower than erythrocyte sedimentation rate in KL-III and -IV (P = 0.012 and 0.037, respectively). Erythrocyte sedimentation rate in KL-II did not significantly differ from erythrocyte sedimentation rate in the other groups. High-sensitivity C-reactive protein concentration was lower in grade I than in KL-II, -III and -IV (P = 0.044, 0.0085 and 0.049, respectively). Conclusions Erythrocyte sedimentation rate and high-sensitivity C-reactive protein concentration were higher in patients with knee osteoarthritis and were related to clinical features. In knee osteoarthritis, high-sensitivity C-reactive protein concentration may increase in early-stage KL-II.

Assessing the validity of the modified Blumensaat method for radiographic evaluation of patellar height.

PurposeAlthough several indexes have been used for patellar height evaluation, the literature on patellar height evaluation using a femoral reference point is scant. We have previously reported on a new index, the modified Blumensaat (MB) method, which evaluates the patellar height using a femoral reference point. The purpose of this study was to evaluate the validity of the MB method.MethodsIn addition to 10 volunteers (group C), 10 men (group M) and 10 women (group F) who underwent knee surgery were selected for the study. The Insall–Salvati (IS), modified IS (MIS), Blackburne–Peel (BP), MB, and modified intercondylar-shelf-angle-corrected Blumensaat (MIB) ratios were measured on lateral knee radiographs with the knee in 30°, 40°, and 50° flexion.ResultsIn group C, the MB and MIB ratios were similar; the IS ratio was not dependent on the knee flexion angle, and there was no significant difference in the MIS and BP ratios between 40° and 50° of knee flexion and in the MB and the MIB ratios between 30° and 40° of knee flexion. There were no differences between group M and group F with respect to any of the indexes. Furthermore, the differences in all indexes among the different knee flexion angles, in groups M and F, were similar to those in group C.ConclusionsThe MB method is applicable for patellar height evaluation and is recommended with a knee flexion angle of 30°–40°.