Harutoshi Sakakima

Different frequency treadmill running in immobilization-induced muscle atrophy and ankle joint contracture of rats

We investigated the effects of different frequencies of treadmill running on immobilization‐induced soleus and gastrocnemius muscle atrophy and ankle joint contracture in rats using morphology and histochemistry. The right ankle joint of rat was immobilized for 2 weeks. Thereafter, the rats were randomly assigned to four groups for 6 weeks of exercise under different conditions: free cage activity and free remobilization (FR), once‐a‐week treadmill running (low‐frequency running program (LFR)), three‐time‐a‐week running (middle‐frequency running program (MFR)), and six‐time‐a‐week running (high‐frequency running program (HFR)) groups. Two weeks of immobilization significantly reduced the cross‐sectional area of soleus type I (62%, P<0.05) and type II muscle fibers (66%, P<0.05), gastrocnemius type I (78%, P<0.05) and type II muscle fibers (68%, P<0.05), and the range of ankle joint movement (46%, P<0.05). Immobilization also increased the ratio of type II to total fiber numbers in the soleus (P<0.05), and gastrocnemius (P<0.05), and induced pathological changes in muscle fibers. Some of these changes could not be corrected by free remobilization; however, the LFR, MFR, and HFR groups clearly recovered toward normal levels with exercise frequency, the effect on muscle recovery being more beneficial in the MFR and HFR groups. In addition, the range of ankle joint contracture was improved in LFR, MFR, and HFR groups in comparison with that in the FR group. These findings indicate that treadmill running exercise improved the immobilization‐induced muscle fiber histochemical alterations and the range of the ankle motion in rats. Running three times and six times a week was more beneficial for recovery of immobilization‐induced muscle atrophy and joint contracture compared with no running or once‐a‐week running.

Accumulation of p62 in degenerated spinal cord under chronic mechanical compression: Functional analysis of p62 and autophagy in hypoxic neuronal cells

Intracellular accumulation of altered proteins, including p62 and ubiquitinated proteins, is the basis of most neurodegenerative disorders. The relationship among the accumulation of altered proteins, autophagy, and spinal cord dysfunction by cervical spondylotic myelopathy has not been clarified. We examined the expression of p62 and autophagy markers in the chronically compressed spinal cord of tiptoe-walking Yoshimura mice. In addition, we examined the expression and roles of p62 and autophagy in hypoxic neuronal cells. Western blot analysis showed the accumulation of p62, ubiquitinated proteins, and microtubule-associated protein 1 light chain 3 (LC3), an autophagic marker, in the compressed spinal cord. Immunohistochemical examinations showed that p62 accumulated in neurons, axons, astrocytes, and oligodendrocytes. Electron microscopy showed the expression of autophagy markers, including autolysosomes and autophagic vesicles, in the compressed spinal cord. These findings suggest the presence of p62 and autophagy in the degenerated compressed spinal cord. Hypoxic stress increased the expression of p62, ubiquitinated proteins, and LC3-II in neuronal cells. In addition, LC3 turnover assay and GFP-LC3 cleavage assay showed that hypoxic stress increased autophagy flux in neuronal cells. These findings suggest that hypoxic stress induces accumulation of p62 and autophagy in neuronal cells. The forced expression of p62 decreased the number of neuronal cells under hypoxic stress. These findings suggest that p62 accumulation under hypoxic stress promotes neuronal cell death. Treatment with 3-methyladenine, an autophagy inhibitor decreased the number of neuronal cells, whereas lithium chloride, an autophagy inducer increased the number of cells under hypoxic stress. These findings suggest that autophagy promotes neuronal cell survival under hypoxic stress. Our findings suggest that pharmacological inducers of autophagy may be useful for treating cervical spondylotic myelopathy patients.

Effects of short duration static stretching on the denervated and reinnervated soleus muscle morphology in the rat.

OBJECTIVE To investigate the effect of short duration static stretching of denervated and reinnervated muscle using a histochemical study on the soleus muscle of the rat. DESIGN Prospective randomized trial. SETTING University medical school in Japan. ANIMALS Fifty-four 8-week-old female Wistar rats with a mean weight +/- standard deviation of 185.8+/-9.9g. INTERVENTIONS After a cold injury was applied to the rat right sciatic nerve, the bilateral soleus muscles were maximally stretched in the dorsiflex posture of the ankle joints for 40 minutes a day, 6 times a week (group S). Rats were compared with nonstretched rats (group D) for up to 4 weeks. Main outcome measures Muscle fiber cross-sectional areas, muscle fiber types, and sciatic nerve morphology. RESULTS No apparent difference in the morphologic changes of the sciatic nerve was found between groups D and S. The mean fiber size progressively declined to a minimum 2 weeks after the injury and reversed in the following weeks. At weeks 1 and 2, the mean type I fiber size in group S was significantly larger than in group D (P<.05). The number ratio of the type II to total fibers increased until 3 weeks in group D, whereas the ratio in group S was increased at 2 weeks, and successively decreased. CONCLUSIONS The mechanical stimuli of static stretching could prevent atrophy of the type I fibers in the denervated muscle and affect the reinnervated muscle fiber-type composition.

A Newly Developed Robot Suit Hybrid Assistive Limb Facilitated Walking Rehabilitation after Spinal Surgery for Thoracic Ossification of the Posterior Longitudinal Ligament: A Case Report

Most patients with thoracic ossification of the posterior longitudinal ligament (OPLL) exhibit delayed recovery of gait dysfunction after spinal injury. The hybrid assistive limb (HAL) is a new robot suit controlling knee and hip joint motion by detecting very weak bioelectric signals on the surface of the skin. This study is to report the feasibility and benefits of patient-assistive HAL walking rehabilitation for facilitating locomotor function after spinal surgery. The patient was a 60-year-old woman with thoracic OPLL, and her motor and sensory paralyses did not improve after spinal surgery, indicating severe impairment in the paretic legs. The subject underwent 6 HAL sessions per week for 8 weeks, consisting of a standing and sitting exercise and walking on the ground with HAL. Clinical outcomes were evaluated before and after HAL training and 1 year after surgery. The subject improved considerably as a result of HAL training. Subsequently, her walking ability recovered rapidly, and she was able to walk unaided six months after surgery. This case study suggests that HAL training is a feasible and effective option to facilitating locomotor function and the early HAL training with physiotherapy may enhance motor recovery of patients with residual paralysis after surgery.

Traumatic Injury-Induced Midkine Expression in the Adult Rat Spinal Cord during the Early Stage

Spinal cord injury is a debilitating condition. Midkine (MK) is involved in the generation of the central nervous system during development; however, the role of MK in the mature spinal cord has not been clarified. We examined the expression of MK, which has neurotrophic activity, before and after traumatic injury to the adult rat spinal cord. Following laminectomy, the rat spinal cord was injured at the T-9 level by applying extradural static weight-compression, in which a cylindrical compressor was used to induce complete and irreversible transverse spinal cord injury with paralysis of the lower extremities. The expression of MK was examined up to 14 days after the injury by immunohistochemical and Western blot analyses. Intense MK immunoreactivity was observed in the gray matter around the injury site but not in the necrotic lesion 1-7 days postinjury, although it was slightly positive 14 days after the injury. MK immunoreactivity was not detected in the normal spinal cord. The expression of MK was an early event, and its expression was compared to the increased production of glial fibrillary acidic protein (GFAP), a marker of reactive astrocytes, that was elevated at 2 days postinjury and continued over a 14 day period following the injury. Double immunostaining with anti-MK and anti-GFAP showed the existence of MK in the astrocytic cytoplasm. These findings suggest that MK was produced in astrocytes approximating the damaged region and may represent a reparative neurotrophic factor during the early phase of traumatic injury of the spinal cord.

Senescence of chondrocytes in aging articular cartilage: GADD45β mediates p21 expression in association with C/EBPβ in senescence-accelerated mice

Growth arrest and DNA damage-inducible protein 45β (GADD45β) is expressed in normal and early osteoarthritic articular cartilage. We recently reported that GADD45β enhances CCAAT/enhancer binding protein β (C/EBPβ) activation in vitro. This study was undertaken in order to determine whether GADD45β is expressed with C/EBPβ in aging articular cartilage. We also investigated whether the synergistic expression of GADD45β and C/EBPβ may be involved in the mechanism of chondrocyte senescence. Senescence-accelerated mice (SAMP1) were used as a model of aging. GADD45β, C/EBPβ, and p21 were analyzed by immunohistochemistry. A luciferase reporter assay using ATDC5 cells was performed in order to examine p21 as a target gene of the GADD45β/C/EBPβ cascade. GADD45β exhibited increased expression in the aging articular cartilage of SAMP1 mice compared to that in control mice. The co-localization of GADD45β and C/EBPβ was confirmed by double immunostaining. The synergistic mechanisms of GADD45β and C/EBPβ on the gene regulation of p21, a molecule related to cellular senescence, were verified by a p21-luciferase reporter assay. Co-expression of C/EBPβ and p21 was confirmed. These observations suggest that the synergism between GADD45β and C/EBPβ may play an important role in cellular senescence in the aging articular cartilage.

The neuroprotective effects of preconditioning exercise on brain damage and neurotrophic factors after focal brain ischemia in rats.

Preconditioning exercise can exert neuroprotective effects after stroke. However, the mechanism underlying these neuroprotective effects by preconditioning exercise remains unclear. We investigated the neuroprotective effects of preconditioning exercise on brain damage and the expression levels of the midkine (MK) and brain-derived neurotrophic factor (BDNF) after brain ischemia. Animals were assigned to one of 4 groups: exercise and ischemia (Ex), no exercise and ischemia (No-Ex), exercise and no ischemia (Ex-only), and no exercise and intact (Control). Rats ran on a treadmill for 30 min once a day at a speed of 25 m/min for 5 days a week for 3 weeks. After the exercise program, stroke was induced by a 60 min left middle cerebral artery occlusion using an intraluminal filament. The infarct volume, motor function, neurological deficits, and the cellular expressions levels of MK, BDNF, GFAP, PECAM-1, caspase 3, and nitrotyrosine (NT) were evaluated 48 h after the induction of ischemia. The infarct volume, neurological deficits and motor function in the Ex group were significantly improved compared to that of the No-Ex group. The expression levels of MK, BDNF, GFAP, and PECAM-1 were enhanced in the Ex group compared to the expression levels in the No-Ex group after brain ischemia, while the expression levels of activated caspase 3 and NT were reduced in the area surrounding the necrotic lesion. Our findings suggest that preconditioning exercise reduced the infract volume and ameliorated motor function, enhanced expression levels of MK and BDNF, increased astrocyte proliferation, increased angiogenesis, and reduced neuronal apoptosis and oxidative stress.

Midkine-deficient mice delayed degeneration and regeneration after skeletal muscle injury

Midkine (MK), a heparin-binding growth factor, was previously found to be expressed in the rat myotube-forming stage. We investigated MK gene-deficient (Mdk(-/-)) mice in terms of skeletal muscle degeneration and regeneration after injury by bupivacaine injection into the tibialis anterior muscle. Injured muscles showed intense inflammatory cell infiltration. Myotubes, myofibers with centrally located nuclei in their cytoplasm, were significantly smaller in Mdk(-/-) mice than in wild type (Mdk(+/+)) mice 7 days after injury (p=0.02). The distribution of myotube sizes showed quantitative differences between the two groups at 5 and 7 days, but not at 14 days. Many small myotubes were found in the regenerative area of Mdk(-/-) mice compared with that of Mdk(+/+)mice 5 and 7 days after injury. The expression of Iba1, a macrophage marker, was significantly lower in Mdk(-/-) mice 3 days after injury (p=0.01). The number of desmin-positive cells like myoblasts in Mdk(-/-) mice was significantly fewer than that in Mdk(+/+) mice 3 days after injury. Our results suggested that deletion of MK results in a delay in regeneration, preceded by decelerated migration of macrophages to the damaged area, and that MK has a role in cell differentiation and maturation after skeletal muscle injury.

Gadd45β expression in chondrosarcoma: A pilot study for diagnostic and biological implications in histological grading

BackgroundAlthough the diagnosis of chondrosarcoma, especially the distinction between enchondroma and low-grade chondrosarcoma or low-grade chondrosarcoma and high-grade chondrosarcoma, is pathologically difficult, differential diagnosis is very important because the treatment strategies for these diseases are completely different. The grading system is crucial in predicting biologic behavior and prognosis, however, exact pathological grading is difficult using only routine examinations because the criteria of the grading system are not necessarily definitive. Growth arrest and DNA damage-inducible protein 45β (GADD45β) is an essential molecule for chondrocytes during terminal differentiation. In the present study, we investigated the immunohistochemical expression of GADD45β in enchondroma, and chondrosarcoma of histological grades I, II, and III, to clarify the diagnostic significance of GADD45β in pathological grading of chondrosarcoma.MethodsTwenty samples (enchondroma = 6, chondrosarcoma grade I = 7, grade II = 6, grade III = 1) were used for immunohistochemical analysis to investigate the expression of GADD45β. Quantitative analysis was performed to compare the number of GADD45β positive cells and pathological grading.ResultsOver 70% of the cells in enchondromas expressed GADD45β. On the other hand, the expression of GADD45β decreased significantly according to the histological grade of chondrosarcoma (grade I: 45%; grade II: 13.8%; and grade III: 3.8%).ConclusionsThe association of GADD45β expression and pathological grading of chondrosarcoma in the present study suggests that the immunohistochemical study of GADD45β may be a specific diagnostic parameter for chondrosarcoma cell differentiation.

Evaluation of occipitocervical subluxation in rheumatoid arthritis patients, using coronal-view reconstructive computed tomography.

Study Design. Reconstructive computed tomography (CT) study of occipito-atlanto and atlantoaxial joints in RA patients. Summary of Background Data. The occipitocervical region is one of the most common sites of rheumatoid arthritis (RA). Although lateral radiography has been used for the diagnosis of atlantoaxial subluxation and vertical subluxation, reconstructive CT imaging of the occipito-atlanto and atlantoaxial joints is more sensitive in detecting morphologic changes in this region. We investigated this region in RA patients, using coronal-view reconstructive CT images, and examined the relationship between the morphology and other radiographic parameters. Methods. The occipitocervical region was examined in 58 female RA patients by reconstructive CT, plain radiography, and MRI. The degree of destructive change on reconstructive CT was compared to that on other radiographic evaluations. Results. Coronal-view reconstructive CT revealed primary destructive changes before detection by lateral radiography, using Redlund-Johnell or Ranawat values. A Redlund-Johnell value less than 34 mm was diagnostic for occipitocervical subluxation in female RA patients. Conclusion. Coronal-view reconstructive CT is useful for the diagnosis of occipitocervical joint subluxation in RA.