Tomoyuki Kurose

Analysis of Gene Expression in Experimental Pressure Ulcers in the Rat with Special Reference to Inflammatory Cytokines.

Pressure ulcers have been investigated in a few animal models, but the molecular mechanisms of pressure ulcers are not well understood. We hypothesized that pressure results in up-regulation of inflammatory cytokines and those cytokines contribute to the formation of pressure ulcers. We measured genome-wide changes in transcript levels after compression, and focused especially on inflammatory cytokines. The abdominal wall of rats was compressed at 100 mmHg for 4 hours by two magnets. Specimens were obtained 12 hours, 1, or 3 days after compression, and analyzed by light microscopy, microarray, Real-Time PCR, and ELISA. The skin and subcutaneous tissue in the compressed area were markedly thickened. The microarray showed that numerous genes were up-regulated after the compression. Up-regulated genes were involved in apoptosis, inflammation, oxidative stress, proteolysis, hypoxia, and so on. Real-Time PCR showed the up-regulation of granulocyte-macrophage colony stimulating factor (GM-CSF), interferon γ (IFN-γ), interleukin 1β (IL-1β), interleukin 1 receptor antagonist gene (IL1Ra), interleukin 6 (IL-6), interleukin 10 (IL-10), matrix metalloproteinase 3 (MMP-3), tissue inhibitor of metalloproteinase 1 (TIMP-1), and tumor necrosis factor α (TNF-α) at 12 hours, IFN-γ, IL-6, IL-10, MMP-3, and TIMP-1 at 1 day, and IFN-γ, IL-6, and MMP-3 at 3 days. Some genes from subcutaneous tissue were up-regulated temporarily, and others were kept at high levels of expression. ELISA data showed that the concentrations of IL-1β and IL-6 proteins were most notably increased following compression. Prolonged up-regulation of IL-1β, and IL-6 might enhance local inflammation, and continuous local inflammation may contribute to the pressure ulcer formation. In addition, GM-CSF, IFN-γ, MMP-3, and TIMP-1 were not reported previously in the wound healing process, and those genes may have a role in development of the pressure ulcers. Expression data from Real-Time PCR were generally in good agreement with those of the microarray. Our microarray data were useful for identifying genes involved in pressure ulcer formation. However, the expression levels of the genes didn’t necessarily correspond with protein production. As such, the functions of these cytokines need to be further investigated.

Interaction between gastrocnemius muscle weakness and moderate exercise deteriorates joint integrity in rat knee

The aim of this study was to determine the effect on the knee joint of the interaction between ankle muscle weakness and moderate exercise. Gastrocnemius muscle weakness was induced by intramuscular injection of botulinum toxin type A (BTX) in rats. Low‐speed treadmill running (12 m/min for 60 min) was applied for 6 weeks in rats with and without BTX. Untreated animals were used as controls. After BTX injection, the gastrocnemius muscle weakness was confirmed by 3‐D motion analysis in kinematic features of the hindlimb during locomotion as an increased maximal dorsiflexion angle during the stance phase. Serum biomarker analysis by enzyme‐linked immunosorbent assay revealed that low‐speed running decreased the catabolic effect on type II collagen. However, the inhibition of catabolism induced by running exercise was significantly counteracted by BTX injection. In addition, thinning of the cartilage layer and a reduction in the chondrocyte density was also found in the tibial plateau of the knee in the BTX‐injected rats after running for 6 weeks. These data suggest that moderate exercise have a positive effect on joint homeostasis. However, ankle muscle weakness may alter the mechanical environment of the knee and impair the integrity of joint cartilage with moderate exercise.

Microvascular circulation at cool, normal and warm temperatures in rat leg muscles examined by histochemistry using Lycopersicon esculentum lectin.

Local cooling and/or warming of the body are widely used for therapy. For safer and more effective therapy, microvascular hemodynamics needs to be clarified. To examine blood circulation in rat leg muscles at 20, 30, 37 and 40°C, fluorescein isothiocyanate (FITC)-labeled Lycopersicon esculentum lectin was injected into the cardiac ventricle. Endothelial cells of open and functioning blood vessels were labeled by this lectin for 3 min and detected by immunostaining for lectin. The percentage of open and functioning capillaries of leg muscles by the avidin-biotin method was 89.8±3.3% at 37°C, while capillaries were unclear or unstained at 20 and 30°C, probably due to a decrease of blood flow. The results using the tyramide-dinitrophenol method were 58.6±15.0% at 20°C, 68.5±12.3% at 30°C, 83.8±5.7% at 37°C and 83.3±7.8% at 40°C. The value at 20°C was significantly different from those at 37 and 40°C. The results by the tyramide-biotin method were 85.5±5.3% at 20°C, 87.3±9.7% at 30°C, 94.7±3.6% at 37°C and 92.5±2.1% at 40°C. Based on these results, it was concluded that the blood flow of each capillary considerably decreased at 20 and 30°C and probably increased at 40°C, whereas the proportion of open and functioning capillaries was essentially unchanged.

Changes in lipid metabolism and capillary density of the skeletal muscle following low-intensity exercise training in a rat model of obesity with hyperinsulinemia

Although exercise is effective in improving obesity and hyperinsulinemia, the exact influence of exercise on the capillary density of skeletal muscles remains unknown. The aim of this study was to investigate the effects of low-intensity exercise training on metabolism in obesity with hyperinsulinemia, focusing specifically on the capillary density within the skeletal muscle. Otsuka Long-Evans Tokushima fatty (OLETF) rats were used as animal models of obesity with hyperinsulinemia, whereas Long-Evans Tokushima Otsuka (LETO) rats served as controls (no obesity, no hyperinsulinemia). The animals were randomly assigned to either non-exercise or exercise groups (treadmill running for 60 min/day, for 4 weeks). The exercise groups were further divided into subgroups according to training mode: single bout (60 min, daily) vs. multiple bout (three bouts of 20 min, daily). Fasting insulin levels were significantly higher in OLETF than in LETO rats. Among OLETF rats, there were no significant differences in fasting glucose levels between the exercise and the non-exercise groups, but the fasting insulin levels were significantly lower in the exercise group. Body weight and triacylglycerol levels in the liver were significantly higher in OLETF than in LETO rats; however, among OLETF rats, these levels were significantly lower in the exercise than in the non-exercise group. The capillary-to-fiber ratio of the soleus muscle was significantly higher in OLETF than in LETO rats; however, among OLETF rats, the ratio was lower in the exercise group than in the non-exercise group. No significant differences in any of the studied parameters were noted between the single-bout and multiple-bout exercise training modes among either OLETF or LETO rats. These results suggest that low-intensity exercise training improves insulin sensitivity and fatty liver. Additionally, the fact that attenuation of excessive capillarization in the skeletal muscle of OLETF rats was accompanied by improvement in increased body weight.