Jiro Nakano

Low‐level laser irradiation promotes the recovery of atrophied gastrocnemius skeletal muscle in rats

Low‐level laser (LLL) irradiation promotes proliferation of muscle satellite cells, angiogenesis and expression of growth factors. Satellite cells, angiogenesis and growth factors play important roles in the regeneration of muscle. The objective of this study was to examine the effect of LLL irradiation on rat gastrocnemius muscle recovering from disuse muscle atrophy. Eight‐week‐old rats were subjected to hindlimb suspension for 2 weeks, after which they were released and recovered. During the recovery period, rats underwent daily LLL irradiation (Ga–Al–As laser; 830 nm; 60 mW; total, 180 s) to the right gastrocnemius muscle through the skin. The untreated left gastrocnemius muscle served as the control. In conjunction with LLL irradiation, 5‐bromo‐2′‐deoxyuridine (BrdU) was injected subcutaneously to label the nuclei of proliferating cells. After 2 weeks, myofibre diameters of irradiated muscle increased in comparison with those of untreated muscle, but did not recover back to normal levels. Additionally, in the superficial region of the irradiated muscle, the number of capillaries and fibroblast growth factor levels exhibited significant elevation relative to those of untreated muscle. In the deep region of irradiated muscle, BrdU‐positive nuclei of satellite cells and/or myofibres increased significantly relative to those of the untreated muscle. The results of this study suggest that LLL irradiation can promote recovery from disuse muscle atrophy in association with proliferation of satellite cells and angiogenesis.

Effects of reduced joint mobility on sarcomere length, collagen fibril arrangement in the endomysium, and hyaluronan in rat soleus muscle.

Immobilization is often associated with a decrease in muscle elasticity. This condition is called muscle contracture, but the mechanism is not yet clear. We examined changes in ankle joint mobility, sarcomere length, collagen fibril arrangement in the endomysium, and hyaluronic acid (HYA) in muscular tissue 1, 2, 4, 8, and 12 weeks after immobilization of rat soleus muscles in shortened position. Ankle joint mobility decreased with the duration of immobilization. Sarcomere length had shortened 1week after immobilization, but did not show further change 2, 4, 8, and 12 weeks after immobilization. Collagen fibril arrangement in the endomysium 1 and 2 weeks after immobilization was longitudinal to the axis of the muscle fibers, whereas 4, 8, and 12 weeks after immobilization it was circumferential. HYA in muscular tissue had increased 1 week after immobilization but remained at the same level at weeks 2, 4, 8, and 12. Histochemically, HYA in the endomysium of immobilized muscular tissue was stained more strongly and widely than that in the control tissue. Increased HYA in muscular tissue may induce muscle stiffness, but the significance of how HYA is related to the mechanism of muscle contracture was not clear. The findings suggest that muscle contracture started 1 week after immobilization and increased with the length of immobilization. Consequently, muscle contracture is affected by the shortening muscle fibers during the early stage of immobilization, after which the collagen adapts by the fibril arrangement in the endomysium becoming more circumferential. This change in collagen fibril arrangement may cause advanced muscle contracture in the late stage of immobilization.

Effects of Therapeutic Ultrasound on Joint Mobility and Collagen Fibril Arrangement in the Endomysium of Immobilized Rat Soleus Muscle

This study examined effects of therapeutic ultrasound on joint mobility and collagen fibril arrangement in the endomysium of immobilized rat soleus muscle. Twenty-two male Wistar rats were divided randomly into control (n = 5) and experimental groups (n = 17). In the experimental group, bilateral ankle joints of each rat were fixed in full plantar flexion with a plaster cast over a 4-wk period. Five animals in the experimental group were immobilized throughout the 4-wk (immobilization group) period, whereas the remaining rats in the experimental group were randomly divided into the ultrasound (US, n = 6) and sham (n = 6) treatment groups. Under anesthesia, continuous ultrasonic energy (frequency, 1 MHz; intensity, 1.0 W/cm(2)) was delivered to the triceps surae muscle of the US group for 15 min per d, 6 d per wk over the 4-wk immobilization period. Ultrasonic energy was not delivered to the triceps surae muscle in sham animals; only the transducer head was moved. Ankle joint mobility on dorsiflexion in the immobilization, sham and US groups was significantly smaller than that of the control group, whereas in the US group, this parameter was significantly greater than in the immobilization and sham groups. Collagen fibril arrangement in the endomysium of the control and US groups was longitudinal to the axis of the muscle fibers; in contrast, it was circumferential in the immobilization and sham groups. Our findings revealed that joint immobilization induces decreased joint mobility and collagen fibril movement in the endomysium; furthermore, ultrasound treatment can prevent these changes. We hypothesized that therapeutic ultrasound during the immobilization process may inhibit deterioration of muscle contracture.

Immobilization-Induced Cartilage Degeneration Mediated Through Expression of Hypoxia-Inducible Factor-1α, Vascular Endothelial Growth Factor, and Chondromodulin-I

Immobilization results in thinning of the articular cartilage and cartilage degeneration, although the exact mechanisms are not clear yet. Hypoxia is thought to contribute to the degeneration of articular cartilage. We investigated the roles of hypoxia inducible factor (HIF)-1α, vascular endothelial growth factor (VEGF), and the newly cloned antiangiogenic factor, chondromodulin-I (ChM-1), in cartilage degeneration in immobilized joints. Male Wistar rats (n = 30, 12-week-old) were divided randomly into the control group (n = 10), immobilization group (n = 10), and continuous passive motion (CPM) group (n = 10). In the immobilization group, the ankle joints were fixed in full plantar flexion with plaster casts for 4 weeks. In the CPM group, the ankle casts were removed during the immobilization period and the ankle joints were subjected to CPM. Significant thinning of the articular cartilage was noted in the immobilization group but not in the control or CPM group. In the immobilized group, vascular channels were found in the area between the calcified cartilage zone and the subchondral bone. The densities of HIF-1α—and VEGF-immunostained cells were higher in the immobilized group than the other two groups. In contrast, low expression of ChM-1 was detected in the articular cartilage of the immobilized group compared with the control and CPM group. Our results showed that immobilization induces thinning of the articular cartilage and appearance of vascular channel, in areas with balanced expression of HIF-1α/VEGF and ChM-1.

Investigation and Macroscopic Anatomical Study of Referred Pain in Patients with Hip Disease

[Purpose] The aim of this study was to examine the incidence and patterns of referred pain in patients with hip disease, as well as the nerve distribution in the hip and knee joints of 2 cadavers. [Subjects and Methods] A total of 113 patients with hip joint disease were included in the investigation. The incidence of regional pain and referred pain patterns were evaluated before and after arthroplasty. Two cadavers were macroscopically observed to verify the nerve innervation of the hip and knee joints. [Results] Anterior knee pain was observed preoperatively in 13.3% (in resting) and 33.6% (in motion) of the patients, which was comparable with the incidence of greater trochanter pain. In addition, the preoperative incidence rates of knee pain in resting and motion markedly decreased postoperatively. Of note is the remarkable incidence of pain radiating to the ventral lower limb. An anteromedial innervation was determined in the cadavers by the articular branches of the obturator and femoral nerve, which supply small branches to the knee joints. [Conclusion] Our results suggest that the distribution of the incidence of pain among the patients with hip disease is diverse owing to the sensory distribution of the femoral and obturator nerves.

Laminin-induced autoimmune myositis in rats.

The present study aimed to examine if immunization with laminin causes myositis in rats and whether the pathologic findings mirror human polymyositis and dermatomyositis. Rats were immunized with an emulsion of laminin and complete Freunds adjuvant. As a result, muscle fiber necrosis with infiltrating macrophages was frequently observed and mononuclear cells were observed in the endomysium. These mononuclear cells were composed of CD4+ cells, CD8+ T cells, and macrophages. CD4+ cells and CD8+ T cells were mainly located in the endomysium, whereas a large number of macrophages were located in the endomysium and infiltrating muscle fibers. A small number of B cells, detected by immunohistochemical staining, were mainly located in the perimysium. The nonnecrotic muscle fiber to which CD4+ T cells, CD8+ T cells, and perforin+ cells adhered was negative for antimerosin and antidystrophin antibodies. Muscle fiber necrosis in rats immunized with laminin may occur after denaturation of basement membrane proteins. In conclusion, the immunization with laminin induces moderate to severe myositis. We suggest that laminin may be an important antigen for connective tissue diseases such as polymyositis and dermatomyositis.

Hyperalgesia in an immobilized rat hindlimb: Effect of treadmill exercise using non-immobilized limbs

Cast immobilization of limbs causes hyperalgesia, which is a decline of the threshold of mechanical and thermal mechanical stimuli. The immobilization-induced hyperalgesia (IIH) can disturb rehabilitation and activities of daily living in patients with orthopedic disorders. However, it is unclear what therapeutic and preventive approaches can be used to alleviate IIH. Exercise that activates the descending pain modulatory system may be effective for IIH. The purpose of this study was to investigate the effects of treadmill exercise during the immobilization period, using the non-immobilized limbs, on IIH. Thirty-six 8-week-old Wistar rats were randomly divided into (1) control, (2) immobilization (Im), and (3) immobilization and treadmill exercise (Im+Ex) groups. In the Im and Im+Ex groups, the right ankle joints of each rat were immobilized in full plantar flexion with a plaster cast for an 8-week period. In the Im+Ex group, treadmill exercise (15 m/min, 30 min/day, 5 days/week) was administered during the immobilization period while the right hindlimb was kept immobilized. Mechanical hyperalgesia was measured using von Frey filaments every week. To investigate possible activation of the descending pain modulatory system, beta-endorphin expression levels in hypothalamus and midbrain periaqueductal gray were analyzed. Although IIH clearly occurred in the Im group, the hyperalgesia was partially but significantly reduced in the Im+Ex group. Beta-endorphin, which is one of the endogenous opioids, was selectively increased in the hypothalamus and midbrain periaqueductal gray of the Im+Ex group. Our data suggest that treadmill running using the non-immobilized limbs reduces the amount of hyperalgesia induced in the immobilized limb even if it is not freed. This ameliorating effect might be due to the descending pain modulatory system being activated by upregulation of beta-endorphin in the brain.

Upregulation of interleukin-1β/transforming growth factor-β1 and hypoxia relate to molecular mechanisms underlying immobilization-induced muscle contracture.

Introduction: In this study we investigated the molecular mechanism underlying muscle contracture in rats. Methods: The rats were divided into immobilization and control groups, and soleus muscles of the right and left sides were selected for analyses. Results: The levels of CD11b and α‐SMA protein, IL‐1β, and TGF‐β1 mRNA, and type I and III collagen protein and mRNA were significantly greater in the immobilization group than in the control group at all time‐points. HIF‐1α mRNA levels were significantly higher in the immobilization group at 4 weeks. Moreover, HIF‐1α, α‐SMA, and type I collagen levels were significantly higher at 4 weeks than at 1 and 2 weeks in the immobilization group. Conclusions: In the early stages of immobilization, upregulation of IL‐1β/TGF‐β1 via macrophages may promote fibroblast differentiation that could affect muscle contracture. The soleus muscle became hypoxic in the later stages of immobilization, suggesting that hypoxia influences the progression of muscle contracture. Muscle Nerve 52:419–427, 2015

Sensory hyperinnervation and increase in NGF, TRPV1 and P2X3 expression in the epidermis following cast immobilization in rats

Cast immobilization is known to induce pain in humans and experimental animal models; however, the detailed mechanisms underlying this pain have yet to be elucidated. Recently, several lines of evidence have indicated that morphological changes in sensory innervation and changes in the expression of pain‐related molecules in the epidermis are related to certain painful conditions. The aim of the present study was to temporally investigate the histological changes in the glabrous skin of the rat hind paw after 1, 2 and 4 weeks of ankle joint immobilization by casting.

Effects of joint immobilization on changes in myofibroblasts and collagen in the rat knee contracture model

The purpose of this study was to examine the time‐dependent changes in the development of joint capsule fibrosis and in the number of myofibroblasts in the joint capsule after immobilization, using a rat knee contracture model. Both knee joints were fixed in full flexion for 1, 2, and 4 weeks (immobilization group). Untreated rats were bred for each immobilization period (control group). Histological analysis was performed to evaluate changes in the amount and density of collagen in the joint capsule. The changes in type I and III collagen mRNA were examined by in situ hybridization. The number of myofibroblasts in the joint capsule was assessed by immunohistochemical methods. In the immobilization group, the amount of collagen increased within 1 week and the density of collagen increased within 2 weeks, as compared with that in the control group. Type I collagen mRNA‐positive cell numbers in the immobilization group increased at all time points. However, type III collagen mRNA‐positive cell numbers did not increase. Myofibroblasts in the immobilization group significantly increased compared with those in the control group at all time points, and they increased significantly with the period of immobilization. These results suggest that joint capsule fibrosis with overexpression of type I collagen occurs and progresses within 1 week after immobilization, and an increase in myofibroblasts is related to the mechanism of joint capsule fibrosis. The findings suggest the need for a treatment targeting accumulation of type I collagen associated with an increase in myofibroblasts.