Ryokei Ogawa

Prevention of senile osteoporosis in SAMP6 mice by intrabone marrow injection of allogeneic bone marrow cells

The SAMP6 mouse (a substrain of senescence‐accelerated mice) spontaneously develops osteoporosis early in life and is, therefore, a useful model for examining the mechanisms underlying osteoporosis. We have recently established a new bone marrow transplantation (BMT) method: the bone marrow cells (BMCs) of normal allogeneic mice are directly injected into the bone marrow (BM) cavity of irradiated (5.5 Gy × 2) recipients (IBM‐BMT). Using IBM‐BMT, we attempted to prevent osteoporosis in SAMP6 mice. The hematolymphoid system was completely reconstituted with donor‐type cells after IBM‐BMT. Thus‐treated SAMP6 mice showed marked increases in trabecular bones even at 12 months of age, and the bone mineral density remained similar to that of normal B6 mice. In concordance with these findings, urinary deoxypyridinoline also remained continuously low until 10 months of age, indicating that IBM‐BMT was effective in the prevention of bone absorption.

Cervical curvature after laminoplasty for spondylotic myelopathy-involvement of yellow ligament, semispinalis cervicis muscle, and nuchal ligament

To assess the consequences of cervical laminoplasty on postoperative lordosis, a retrospective radiographic analysis of 31 patients undergoing laminoplasty for spondylotic myelopathy was completed. Special attention was paid to lordotic changes occurring at each level over more than 2 years. Preoperative lordosis remained unchanged with the patients wearing a cervical orthosis 1 week postoperatively. However the lordosis subsequently demonstrated a significant decrease in 87% of patients over an average of 3.1 years. Lordotic alignment at C2-C3 and C6-C7 before surgery significantly decreased in 81% and 58% of patients 1 week postoperatively, and 84% and 81% at last follow up, respectively, while lordotic alignment at other levels pre- and postoperatively did not significantly change. Loss of lordotic alignment was largely attributed to detachment of semispinalis cervicis muscle on C2 and nuchal ligament on C6/C7 with a posterior approach and/or section of yellow ligament at C2-C3.

Newly designed computer controlled knee-ankle-foot orthosis (Intelligent Orthosis)

The authors have developed a knee-ankle-foot orthosis with a joint unit that controls knee movements using a microcomputer (Intelligent Orthosis). The Intelligent Orthosis was applied to normal subjects and patients, and gait analysis was performed. In the gait cycle, the ratio of the stance phase to the swing phase was less in gait with the knee locked using a knee-ankle-foot orthosis than in gait without an orthosis or gait with the knee controlled by a microcomputer. The ratio of the stance phase to the swing phase between controlled gait and normal gait was similar. For normal subjects the activity of the tibialis anterior was markedly increased from the heel-off phase to the swing phase in locked gait. The muscle activities of the lower limb were lower in controlled force in locked gait showed spikes immediately after heel-contact in the vertical at heel-contact in the sagittal to locked gait, gait with the Intelligent Orthosis is smooth and close to normal gait from the viewpoint of biomechanics. Even in patients with muscle weakness of the quadriceps, control of the knee joint using the Intelligent Orthosis resulted in a more smooth gait with low muscle discharge.

Localization of protein disulfide isomerase on plasma membranes of rat exocrine pancreatic cells.

We investigated immunocytochemically the ultrastructural localization of protein disulfide isomerase (PDI) in rat pancreatic exocrine cells by use of the post-embedding protein A-gold technique. We found that not only the endoplasmic reticulum (ER) and nuclear envelope but also the trans-Golgi cisternae, secretory granules, and plasma membranes were heavily labeled with gold particles. Labeling density of the gold particles in the rough ER and plasma membranes of the exocrine pancreatic cells was twofold and twentyfold greater, respectively, than that of hepatocytes. In the acinar lumen, amorphous material presumably corresponding to the secreted zymogens was also labeled with gold particles. These results suggest that in rat exocrine pancreatic cells a significant amount of PDI is transported to the plasma membrane and secreted to the acinar lumen.

A New Method for Bone Marrow Cell Harvesting

To minimize contamination of bone marrow cells (BMCs) with T cells from the peripheral blood, a new “perfusion method” for collecting BMCs is proposed using cynomolgus monkeys. Two BM puncture needles are inserted into a long bone such as the humerus, femur, or tibia. One needle is connected to an extension tube and the end of the tube is inserted into a culture flask to collect the BM fluid. The other needle is connected to a syringe containing 30 ml of phosphate‐buffered saline. The solution is pushed gently from the syringe into the medullary cavity, and the medium containing the BM fluid is collected into the culture flask. There is significantly less contamination with peripheral blood, determined from the frequencies of CD4+ and CD8+ T cells, when using this method (<6%) than when using the conventional method (>20%) consisting of multiple BM aspirations from the iliac crest. Furthermore, the number and progenitor activities of the cells harvested using this “perfusion method” are greater than those harvested using the conventional aspiration method. This perfusion method was carried out 42 times using 15 cynomolgus monkeys, and no complications such as pulmonary infarction or paralysis were observed. These findings suggest that the “perfusion method” is safe and simple and would be of great advantage in obtaining pure BMCs, resulting in a less frequent occurrence of acute graft‐versus‐host‐disease in allogeneic BM transplantation.

Crucial Role of Donor‐Derived Stromal Cells in Successful Treatment for Intractable Autoimmune Diseases in MRL/lpr Mice by BMT Via Portal Vein

We have recently established a new bone marrow transplantation (BMT) method for the treatment of intractable autoimmune diseases in MRL/lpr mice; the method consists of fractionated irradiation (5.5 Gy × 2), followed by BMT of whole bone marrow cells (BMCs) from allogeneic C57BL/6 mice via the portal vein (abbreviated as 5.5 Gy × 2 + PV). In the present study, we investigate the mechanisms underlying the early engraftment of donor‐derived cells in MRL/lpr mice by this method. In the mice treated with this method, the number of donor‐derived cells possessing the mature lineage (Lin) markers rapidly increased in the BM, spleen, and liver; almost 100% were donor‐derived cells by 14 days after the treatment. The number of donor‐derived hemopoietic progenitor cells (defined as c‐kit+/Lin− cells) increased in the BMCs, hepatic mononuclear cells, and especially spleen cells by 14 days after the treatment. Simultaneously, hemopoietic foci adjoining donor‐derived stromal cells were observed in the liver when injected via the PV, but not via the peripheral vein (i.v.). When adherent cell‐depleted BMCs were injected via the PV, recipients showed a marked reduction in the survival rate. However, when mice were transplanted with adherent cell‐depleted BMCs with cultured stromal cells, all the recipients survived.

Distribution of protein disulfide isomerase in rat hepatocytes.

We investigated quantitatively the distribution of protein disulfide isomerase (PDI) in rat hepatocytes by immunocytochemistry using a post-embedding protein A-gold technique. In hepatocytes, gold particles were mainly localized in the intracisternal space of the rough and smooth endoplasmic reticulum (ER) and nuclear envelopes. Autolysosomes engulfing ER were occasionally densely labeled, especially in rat hepatocytes previously treated with leupeptin in vivo, suggesting that the autophagosome-autolysosome system may be an important route for degradation of PDI. A few gold particles were also found on the plasma membranes. Localization of gold particles on the other subcellular organelles, such as Golgi apparatus, peroxisomes, and nuclear matrix, was sparse and at the control level. The predominant localization of PDI on the intracisternal surface of the ER and nuclear envelope supports a potential role of PDI in the formation of disulfide bonds of nascent polypeptides, thus accelerating formation of the higher-order structure of secretory and membrane proteins and rendering the translocation process irreversible.

Transcranial magnetic stimulation of the motor cortex in cervical spondylosis and spinal canal stenosis

Study Design. This study investigated the clinical usefulness of motor evoked potentials and a silent period after motor evoked potentials produced by transcranial magnetic stimulation of the brain. Objective. The results were correlated with the clinical state of the patients with myelopathy, whereas no abnormality of the conduction time was observed in the patients with spinal canal stenosis. Summary of Background Data. Magnetic stimulation has been widely used for examination of the descending excitatory motor pathways in the central nervous system, but little attention has been paid to cervical spondylosis and spinal canal stenosis. Methods. Motor evoked potentials were examined in 35 normal subjects, 67 patients with cervical spondylotic myelopathy, and 24 patients with spinal canal stenosis. Motor evoked potentials were evoked by transcranial brain stimulation during relaxation and during maximum voluntary contraction of the target muscle. Results. The central motor conduction time was found to correlate with the clinical state of the myelopathy patients, whereas no abnormality of the conduction time was observed in the patients with spinal canal stenosis. During maximum voluntary contraction of the target muscle, a silent period was always observed after the motor evoked potentials in the normal subjects, and its duration was markedly shortened in the myelopathy patients. Conclusions. In cervical myelopathy patients, the central motor conduction time was correlated with clinical evaluation and the silent period was significantly shortened. These findings about duration of the central motor conduction time and the silent period might be a useful parameter of spinal pathology.

The homozygote of HLA-DRB1*0901, not its heterozygote, is associated with rheumatoid arthritis in Japanese

To assess the association between HLA-DRB1*0901 and Japanese rheumatoid arthritis (RA) patients, we analyzed the frequency of HLA-DRB1*0901 in 852 Japanese RA patients. We found that the homozygote of DRB1*0901 was associated with Japanese RA patients, while the heterozygote of DRB1*0901 was not. These findings suggest that DRB1*0901 is a weakly susceptible allele of RA, which in our investigation was not associated with RA by a single allele, but can be by a homozygote. DRB1*0901 does not have the shared epitope, and it is suggested that there may be some mechanism ofthe association between HLA-DRB1 and RA other than the shared epitope, which was not strong.

The long-term results of ankle joint synovectomy for rheumatoid arthritis

SummaryThe clinical and radiological results of synovectomy for rheumatoid arthritis in the ankle joint were investigated in 20 ankles of 15 patients. The average follow-up period after synovectomy was 15 years, ranging from 10 to 25 years. The clinical evaluation at the time of follow-up, found that only two ankles showed recurrence of synovitis, and no patient complained of severe ankle pain disturbing the activities of daily life. During the period between the synovectomy and our investigation, no patients required further surgical procedures for their ankle joints. The radiological evaluation found that in approximately two-thirds of the cases, deterioration of the radiological grade, evaluated with Larsens criteria, had continued after synovectomy. There was no considerable radiological deterioration in the less-erosive subset patients, classified according to Ochis criteria (1). In the unilateral synovectomized cases, using the non-operated ankles as the natural-course control, osteoarthritic changes were predominant in the operated ankle joint, and the non-operated ankle demonstrated inflammatory disease changes. These results indicate that: (1) synovectomy for a rheumatoid ankle is still a preferred treatment, lessening the clinical symptom of persistent, marked synovial proliferation resistant to medical treatment. (2) Radiological deterioration continues after synovectomy in many cases. However, a radiogram demonstrates predominant osteoarthritic destruction, which indicates the natural course of rheumatoid destruction in the operated site could be altered by synovectomy.