Effects of the magnitude of pressure on the severity of injury and capillary closure in rat experimental pressure ulcers.

Experimental pressure ulcers were successfully produced in the rat abdominal wall at 100 mmHg in our previous study. We hypothesized that injury is less severe when pressures are lower than 100 mmHg and explored a critical pressure in the production of pressure ulcers. At 70 and 60 mmHg, repeated compressions for 4 h daily for 5 consecutive days resulted in partial skin necrosis and eschar formation in the majority of rats, whereas skin injuries were absent or very mild in most of the rats at 50 mmHg. The extent of ischemia was also examined by visualization of capillary blood flow using intravascular infusion of Lycopersicon esculentum lectin. Rat abdominal walls were compressed in the range from 0 (control) to 100 mmHg. The percentages of open capillaries were 62.8 ± 10.1% at 0 mmHg and 34.7 ± 18.5% at 10 mmHg. The ratio of open capillaries was further decreased with increasing pressure, but not pressure dependently. In conclusion, the severity of injury at 50 mmHg was drastically milder than that at 60 mmHg or higher, whereas the extent of ischemia (capillary closure) was not significantly different. The pressure is vitally important; however, other factor(s) besides ischemia is likely to promote the development of pressure ulcers.

Intermittent whole-body vibration attenuates a reduction in the number of the capillaries in unloaded rat skeletal muscle.

BACKGROUND: Whole-body vibration has been suggested for the prevention of muscle mass loss and muscle wasting as an attractive measure for disuse atrophy. This study examined the effects of daily intermittent whole-body vibration and weight bearing during hindlimb suspension on capillary number and muscle atrophy in rat skeletal muscles. METHODS: Sixty male Wistar rats were randomly divided into four groups: control (CONT), hindlimb suspension (HS), HS + weight bearing (WB), and HS + whole-body vibration (VIB) (n = 15 each). Hindlimb suspension was applied for 2 weeks in HS, HS + WB, and HS + VIB groups. During suspension, rats in HS + VIB group were placed daily on a vibrating whole-body vibration platform for 20 min. In HS + WB group, suspension was interrupted for 20 min/day, allowing weight bearing. Untreated rats were used as controls. RESULTS: Soleus muscle wet weights and muscle fiber cross-sectional areas (CSA) significantly decreased in HS, HS + WB, and HS + VIB groups compared with CONT group. Both muscle weights and CSA were significantly greater in HS + WB and HS + VIB groups compared with HS group. Capillary numbers (represented by capillary-to-muscle fiber ratio) were significantly smaller in all hindlimb suspension-treated groups compared with CONT group. However, a reduction in capillary number by unloading hindlimbs was partially prevented by whole-body vibration. These findings were supported by examining mRNA for angiogenic-related factors. Expression levels of a pro-angiogenic factor, vascular endothelial growth factor-A mRNA, were significantly lower in all hindlimb suspension-treated groups compared with CONT group. There were no differences among hindlimb suspension-treated groups. Expression levels of an anti-angiogenic factor, CD36 (receptor for thrombospondin-1) mRNA, were significantly higher in all hindlimb suspension-treated groups compared with CONT group. Among the hindlimb suspension-treated groups, expression of CD36 mRNA in HS + VIB group tended to be suppressed (less than half the HS group). CONCLUSIONS: Our results suggest that weight bearing with or without vibration is effective for disuse-derived disturbance by preventing muscle atrophy, and whole-body vibration exercise has an additional benefit of maintaining microcirculation of skeletal muscle.

Enhanced anxiety-like behavior induced by chronic neuropathic pain and related parvalbumin-positive neurons in male rats.

Anxiety commonly co-occurs with and exacerbates pain, but the interaction between pain progression and anxiety, and its underlying mechanisms remain unclear. Inhibitory interneurons play a crucial role in maintaining normal central nervous system function and are suggested to be involved in pain-induced anxiety. This study aimed to elucidate the time-dependent effects of neuropathic pain on the developmental anxiety-like behaviors and related inhibitory interneurons; parvalbumin (PV)- and cholecystokinin (CCK)-positive neurons in corticolimbic regions. Using an 8-week-old male Wistar rat model with partial sciatic nerve ligation (pSNL), anxiety-like behaviors were biweekly assessed post-surgery through open field (OF) and elevated plus maze (EPM) tests. From 4 weeks post-surgery, pSNL rats exhibited reduced OF center time, rearing, and initial activity, along with diminished EPM open-arm activities (time spent, head dips, movement, and rearing), which correlated with the paw withdrawal threshold. These effects were absent at 2 weeks post-surgery. At 8 weeks post-surgery, specific behaviors (decreased total rearing and increased inactive time in EPM) were observed in the pSNL group. Immunohistochemistry revealed changes in PV- and CCK-positive neurons in specific corticolimbic subregions of pSNL rats at 8 weeks post-surgery. Notably, PV-positive neuron densities in the basolateral amygdaloid complex (BLC) and hippocampal cornu ammonis areas 1 and 2 correlated with anxiety-like behavioral parameters. PV-positive neurons in the BLC of pSNL rats were predominantly changed in large-cell subtypes and were less activated. These findings indicate that anxiety-like behaviors emerge in the late phase of neuropathic pain and relate to PV-positive neurons in corticolimbic regions of pSNL rats.

Hypergravity enhances RBM4 expression in human bone marrow-derived mesenchymal stem cells and accelerates their differentiation into neurons.

Introduction: The regulation of stem cell differentiation is important in determining the quality of transplanted cells in regenerative medicine. Physical stimuli are involved in regulating stem cell differentiation, and in particular, research on the regulation of differentiation using gravity is an attractive choice. We have shown that microgravity is useful for maintaining undifferentiated mesenchymal stem cells (MSCs). However, the effects of hypergravity on the differentiation of MSCs, especially on neural differentiation related to neural regeneration, have not been elucidated. Methods: We induced neural differentiation of human bone marrow-derived MSCs (hbMSCs) for 10 days under normal gravity (1G) or hypergravity (3G) conditions using a gravity controller, Gravite®. HbMSCs were collected, and cell number and viability were measured 3 and 10 days after induction. RNA was also extracted from the collected hbMSCs, and the expression of neuron-associated genes and regulator markers of neural differentiation was analyzed using real-time polymerase chain reaction (PCR). Additionally, we evaluated the NF-M-positive cell rate 10 days after induction using immunofluorescent staining. Results: Neural gene expression and the NF-M-positive cell rate were increased in hbMSCs under the 3G condition 10 days after induction. mRNA expression of RNA binding motif protein 4 (RBM4) and pyruvate kinase M 1 (PKM1) in the 3G condition was also higher than that in the 1G group. Conclusions: Hypergravity can enhance RBM4 and PKM1, promoting the neural differentiation of hbMSCs.

Comparisons of Neurotrophic Effects of Mesenchymal Stem Cells Derived from Different Tissues on Chronic Spinal Cord Injury Rats.

Cell-based therapies with mesenchymal stem cells (MSCs) are considered as promising strategies for spinal cord injury (SCI). MSCs have unique characteristics due to differences in the derived tissues. However, relatively few studies have focused on differences in the therapeutic effects of MSCs derived from different tissues. In this study, the therapeutic effects of adipose tissue-derived MSCs, bone marrow-derived MSCs, and cranial bone-derived MSCs (cMSCs) on chronic SCI model rats were compared. MSCs were established from the collected adipose tissue, bone marrow, and cranial bone. Neurotrophic factor expression of each MSC type was analyzed by real-time PCR. SCI rats were established using the weight-drop method and transplanted intravenously with MSCs at 4 weeks after SCI. Hindlimb motor function was evaluated from before injury to 4 weeks after transplantation. Endogenous neurotrophic factor and neural repair factor expression in spinal cord (SC) tissue were examined by real-time PCR and western blot analyses. Although there were no differences in the expression levels of cell surface markers and multipotency, expression of Bdnf, Ngf, and Sort1 (Nt-3) was relatively higher in cMSCs. Transplantation of cMSCs improved motor function of chronic SCI model rats. Although there was no difference in the degree of engraftment of transplanted cells in the injured SC tissue, transplantation of cMSCs enhanced Bdnf, TrkB, and Gap-43 messenger RNA expression and synaptophysin protein expression in injured SC tissue. As compared with MSCs derived other tissues, cMSCs highly express many neurotrophic factors, which improved motor function in chronic SCI model rats by promoting endogenous neurotrophic and neural plasticity factors. These results demonstrate the efficacy of cMSCs in cell-based therapy for chronic SCI.

Early Transplantation of Human Cranial Bone-derived Mesenchymal Stem Cells Enhances Functional Recovery in Ischemic Stroke Model Rats.

We analyzed the cell characteristics, neuroprotective, and transplantation effects of human cranial bone-derived mesenchymal stem cells (hcMSCs) in ischemic stroke model rats compared with human iliac bone-derived mesenchymal stem cells (hiMSCs). The expressions of brain-derived neurotrophic factor (BDNF) and vascular endothelial growth factor (VEGF ) as neurotrophic factors were analyzed in both MSCs. hiMSCs or hcMSCs were intravenously administered into ischemic stroke model rats at 3 or 24 h after middle cerebral artery occlusion (MCAO) and neurological function was evaluated. The survival rate of neuroblastoma × glioma hybrid cells (NG108-15) after 3 or 24 h oxidative or inflammatory stress and the neuroprotective effects of hiMSCs or hcMSCs-conditioned medium (CM) on 3 or 24 h oxidative or inflammatory stress-exposed NG108-15 cells were analyzed. The expressions of BDNF and VEGF were higher in hcMSCs than in hiMSCs. hcMSCs transplantation at 3 h after MCAO resulted in significant functional recovery compared with that in the hiMSCs or control group. The survival rate of stress-exposed NG108-15 was lower after 24 h stress than after 3 h stress. The survival rates of NG108-15 cells cultured with hcMSCs-CM after 3 h oxidative or inflammatory stress were significantly higher than in the control group. Our results suggest that hcMSCs transplantation in the early stage of ischemic stroke suppresses the damage of residual nerve cells and leads to functional recovery through the strong expressions of neurotrophic factors. This is the first report demonstrating a functional recovery effect after ischemic stroke following hcMSCs transplantation.

Simulated microgravity-cultured mesenchymal stem cells improve recovery following spinal cord ischemia in rats.

Spinal cord ischemia is a potential complication of thoracoabdominal aortic surgery that may induce irreversible motor disability. We investigated the therapeutic efficacy of simulated microgravity-cultured mesenchymal stem cell (MSC) injection following spinal cord ischemia-reperfusion injury. Sprague-Dawley rats were divided into sham, phosphate-buffered saline (PBS), normal gravity-cultured MSC (MSC-1 G), and simulated microgravity-cultured MSC (MSC-MG) groups. Spinal cord ischemia was induced by transient balloon occlusion of the thoracic aorta, which was followed immediately by PBS or MSC injection into the left carotid artery. Hindlimb motor function was evaluated by the Basso-Beattie-Bresnahan (BBB) scale. Spinal cords were removed 1, 3, or 7 days post-injury for immunohistochemical staining and Western blot analysis. One day post-injury, a few infiltrating inflammatory cells and small vacuoles were observed without significant group differences, followed over several days by progressive spinal cord degeneration. Glial fibrillary acidic protein (GFAP)-positive (reactive) astrocyte numbers were increased in all three groups, and brain-derived neurotrophic factor (BDNF) was colocalized with GFAP-positive cells in spinal ventral horn. Animals in the MSC-MG group demonstrated greater BDNF-positive astrocyte numbers, reduced caspase-3-positive cell numbers, and superior motor recovery. Microgravity-cultured MSC-based therapy may improve functional recovery following spinal ischemia-reperfusion injury by promoting astrocytic BDNF release, thereby preventing apoptosis.

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.

Distribution and change of collagen types I and III and elastin in developing leg muscle in rat.

The distribution of collagen types I and III and elastin in the developing leg muscles were studied by immunohistochemistry in rat. From 0-day to 8-weeks old, the size of the gastrocnemius and plantaris muscles increased. The muscle connective tissue developed in the order of epimysium, perimysium and finally endomysium. The epimysium contained a considerable amount of collagen types I and III and some elastin in the neonates. These components in the epimysium remained almost unchanged in their distribution during development. The perimysium had little collagen type I and III or elastin at 0 day. Collagen type I and elastin slightly increased around 2 and 1 week, respectively, and returned to the previous levels. Collagen type III, however, increased and became abundant after 1 week. In the endomysium, the amounts of collagen type I and elastin were slight during postnatal growth, while collagen type III gradually increased after 2 weeks. The intramuscular tendons consistently showed intense reactivity for collagen type I and weak staining for elastin, whereas the staining for collagen type III decreased after 1 week and was finally restricted to the surface of intramuscular tendons. This study clearly demonstrated that the distribution of collagens, but not of elastin, significantly changed during development. The increase in collagen type III in the perimysium and endomysium, and its decrease in the intramuscular tendons probably reflect functional demands imposed on these connective tissues, i.e., shear forces in the former two and tensile loading in the latter.

Regulation of the microvascular circulation in the leg muscles, pancreas and small intestine in rats.

To study the microvascular circulation, we examined the proportion of open and functioning capillaries in the leg muscles, pancreas and small intestine of anesthetized rats. Fluorescein isothiocyanate (FITC)-labeled Lycopersicon esculentum lectin was injected into the heart and allowed to circulate for 3 min to label open and functioning capillaries. Specimens were removed, frozen, sectioned and double-immunostained. Using one section, open and functioning capillaries were detected by immunostaining for this lectin bound to endothelial cells, while all capillaries were visualized by immunostaining for platelet endothelial cell adhesion molecule-1 (PECAM-1 or CD31). These capillaries were semi-automatically detected and counted by fluorescence microscopy. The percentages of open and functioning capillaries were as follows: the soleus muscle, 93.0 ± 5.5%; superficial zone of the gastrocnemius muscle, 90.8 ± 6.2%; deep zone of the gastrocnemius muscle, 95.6 ± 4.0%; the plantaris muscle, 94.1 ± 2.7%; the pancreas, 86.3 ± 11.7%; and the small intestine, 91.1 ± 4.9% (n = 8, each). There was no significant difference among these data by the Kruskal-Wallis test. This study clearly demonstrated that the proportions of open and functioning capillaries are high and similar among the leg muscles, pancreas and small intestine in spite of their structural and functional differences. This finding agrees with previous studies and supports the notion that the microvascular circulation is mainly controlled by changing of the blood flow in each capillary rather than changing the proportion of open and functioning capillaries.

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 1ß (IL-1ß), 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-1ß and IL-6 proteins were most notably increased following compression. Prolonged up-regulation of IL-1ß, 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.

Formaldehyde concentration in the air and in cadavers at the gross anatomy laboratory in Hiroshima University.

The formaldehyde concentration in the air and in various tissues of 35 human cadavers were measured during a gross anatomy course held at the Faculty of Medicine of Hiroshima University in the 2003 educational year. Atmospheric formaldehyde levels were 0.25-0.55 ppm and thus less than the upper limit of the guideline for formaldehyde exposure (0.5 ppm) set by the Japan Society for Occupational Health (1988) except for one out of 10 measurements. The formaldehyde concentrations in tissues were as follows: the lung, 0.12 +/- 0.09% (n=29); the liver, 0.12 +/- 0.09% (n=29); and the brachioradialis muscle, 0.11 +/- 0.09% (n=30). Considerable variation was found among the cadavers and these values were lower than those of Tsurumi University which provided the only other data (average formaldehyde concentrations ranged from 0.27 to 0.32%). At Hiroshima University, blood is allowed to drain during embalming, whereas it is not at Tsurumi University. Differences in the embalming procedure are thus responsible for low and fluctuating formaldehyde concentrations in cadavers at Hiroshima University, and it is conceivable that relatively low formaldehyde levels in the air result from low formaldehyde concentrations in cadavers and good room ventilation (10 room-air changes per hour). However, the Japanese Ministry of Health and Welfare recommended lower formaldehyde exposure levels (0.08 or 0.25 ppm) in 2002. Thus, it may be necessary to further reduce formaldehyde levels in the gross anatomy laboratory by means of such measures as neutralizing formaldehyde with ammonium carbonate; using a locally ventilated dissection work-table, etc.

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.

Regulation of connective tissue remodeling in the early phase of denervation in a rat skeletal muscle.

Denervation alters the metabolism of the extracellular matrix (ECM) in skeletal muscle; however, the underlying mechanisms of ECM remodeling are not fully understood. The aim of this study was to elucidate the dynamic features of the ECM regulatory process in the early phase of denervated skeletal muscle in male Wistar rats. We investigated the expression of collagens (total, type I, and type III), transforming growth factor beta 1 (TGF-ß1), and matrix metalloproteases (MMPs) together with their endogenous inhibitors (TIMPs), at the mRNA and/or protein level in the soleus muscles of control animals and at days 3, 7, and 14 post-denervation. Expression of mRNA encoding collagens was decreased at days 3 and 7, and had recovered by day 14, in parallel with total collagen protein content. Content of TGF-ß1 protein was elevated sequentially, up to a maximum of 158% at day 14 post-denervation (P < 0.05), as was TIMP-2 mRNA expression (272% at day 14), whereas MMP-1, MMP-2, and TIMP-1 mRNA expression was not affected at any stage. The initial reduction of collagen mRNA may be responsible for hypoactivity caused by the disappearance of contractile function. Recovery of collagen mRNA/protein at day 14 may be due mainly to the suppressive effects of TGF-ß1 on collagen degradation via TIMP-2 upregulation.

Reduced soleus muscle injury at long muscle length during contraction in the rat.

Muscle injury was studied to test the hypotheses that maintaining the soleus muscle at a long muscle length during contraction prevents muscle injuries and that the prevention of initial muscle injuries reduces subsequent muscle damage. The rat sciatic nerve was stimulated for 30 min with plantar or dorsal flexion of the foot, and the time course of contraction-induced injuries was examined. The soleus muscle injuries were first classified into one of five types, and the percentages of aberrant sarcomere areas observed in the soleus muscle were then separately quantified by electron microscopy at 0, 1, 6, 12, and 24 h (n = 3) post-stimulation. At a short muscle length (plantar flexion) during contraction, the soleus muscle showed sarcomere hypercontraction (9.8 ± 2.5%, mean ± standard error) and Z-band disarrangement (31.0 ± 4.5%) at 0 h, sarcomere hypercontraction (6.7 ± 1.9%), Z-band disarrangement (28.0 ± 4.9%), and sarcomere hyperstretching (1.3 ± 1.3%) at 1 h, the absence of sarcomere hypercontraction, but Z-band disarrangement (6.7 ± 1.9%) and sarcomere hyperstretching (5.0 ± 1.8%) at 6 h, and myofilament disorganization at 12 and 24 h (5.2 ± 1.5 and 2.5 ± 1.0%, respectively). In contrast, the soleus muscles at a long muscle length (dorsal flexion) during contraction using a self-made brace showed alterations in 1.2-2.4% of sarcomeres at 0 h and afterwards. Desmin disappeared, and α-actinin immunostaining was weaker in areas of sarcomere hypercontraction, whereas dystrophin was always detected along the sarcoplasmic membrane, suggesting that the integrity of the sarcolemma was intact. These results indicate that initial and subsequent muscle injuries were significantly reduced at long muscle length during contraction, probably through the prevention of sarcomere hypercontraction, and that initial muscle injuries rapidly progress to other injuries or normal structure.

Comparison between a weight compression and a magnet compression for experimental pressure ulcers in the rat. Histological studies and effects of anesthesia.

To develop an experimental model and evaluate the effects of the magnitude and duration of pressure, the rat abdominal wall (25x20 mm) was subjected to compression either by a weight or by two magnets. In the weight compression tests, a steel plate was inserted under anesthesia into the rat peritoneal cavity, and the abdominal wall was compressed in situ between the underlying steel plate and a weight placed on the abdominal wall. This method resulted in moderate changes in the subcutaneous connective tissue and muscle at 100 mmHg (13.3 kPa) for 4 h, while some muscle damage was observed at 50 mmHg (6.7 kPa) for 4 h and at 100 mmHg for 2 or 3 h. In the magnet pinching tests, a magnet was inserted into the peritoneal cavity, and another magnet overlaid on the skin. Then the abdominal wall was compressed by the two magnets with or without anesthesia. The compression without anesthesia produced significant edema and injuries of the abdominal wall at 50 mmHg for 4 h and at 100 mmHg for 3 or 4 h, while the injuries incurred at 100 mmHg for 2 h were mild. Susceptibility to pressure was high in the muscle, moderate in the subcutaneous connective tissue, and low in the skin. The compression with anesthesia produced significantly milder injuries than those under anesthesia. These findings indicate that the difference in the extent of injuries between the weight compression and magnet compression models are clearly attributable to the pentobarbital anesthesia induced during the compression. Results therefore show that experimental pressure ulcers should be examined in a waking condition and that magnet compression is a useful model for studying the pathogenesis of pressure ulcers.

The skeletal muscle vascular supply closely correlates with the muscle fiber surface area in the rat.

The skeletal muscle capillary supply (capillarity) dynamically changes in response to muscle conditions such as growth, atrophy, and hypertrophy. The capillary number-to-fiber ratio is reported to correlate closely with the muscle fiber cross sectional area. However, little information is available regarding the capillarity of neonatal and very young skeletal muscles. In this study, the vascular endothelium was reliably stained with an anti-PECAM-1 antibody, and relationships between the capillarity and muscle fiber parameters were analyzed. For assessment of the capillarity, we used the capillary length-to-fiber ratio, due to the presence of transversely running vessels. In young and adult rats, the capillary length-to-fiber ratio was proportional to both the muscle fiber cross sectional area and muscle fiber radius. However, when these data were analyzed together with data from neonatal and very young rats, the capillary length-to-fiber ratio correlated more closely with the muscle fiber radius than the muscle fiber cross sectional area in the tibialis anterior muscle. The capillary number-to-fiber ratio demonstrated results very similar to the capillary length-to-fiber ratio. During muscle atrophy after denervation, the number of capillaries was decreased in a non-apoptotic manner as revealed by electron microscopy, maintaining the close relationship between the parameters described above. In conclusion, capillarity was closely correlated with the muscle fiber radius (which represents the perimeter) during growth and atrophy. This indicates that the capillarity is linked to the muscle fiber surface area (which is determined by perimeter and section thickness), in agreement with the essential role of the cell membrane in the transport of materials by simple diffusion or active transport.

Changes in the rat subcutaneous connective tissue after saline and histamine injection in relation to fluid storage and excretion.

An experimental design was developed for morphometric analysis of the subcutaneous connective tissue after the subcutaneous injection of 0.1 ml of saline or a histamine solution (0.01, 0.1 or 1% histamine dihydrochloride in saline). The subcutaneous connective tissue of 4-week-old rats, originally 170.0 +/- 13.6 mum in thickness, swelled 5.2-fold at 15 min, 3.0-fold at 2 h, and 1.2-fold at 6 h after the injection of saline. The total cross sectional area of both blood and lymphatic vessels increased when compared to that at pre-injection (0.0186 +/- 0.0030 mm(2) in 6-mm-long specimen), 1.4-fold at 15 min, 2.2-fold at 2 h, and 1.1-fold at 6 h post-injection, while the total number of these vessels increased 1.1-fold at 2 h. The total cross sectional area of lymphatic vessels (0.0006 +/- 0.0002 mm(2) in 6-mm-long specimen) alone surged 7.7-fold at 15 min, 4.8-fold at 2 h, and 7.3-fold at 6 h. Collagen fibers were respectively highly, moderately, and mildly disorganized in arrangement at 15 min, 2 h, and 6 h after the saline injection. Histamine elicited an earlier, longer, and more pronounced vasodilatation, particularly at high concentrations. The transvascular permeability of Evans blue increased depending on the concentration of histamine. These findings indicate that the subcutaneous connective tissue has the ability to expand and store a considerable amount of fluid and reversibly returns to normal steady-state conditions by increasing fluid excretion into the blood and lymphatic vessels. It was also strongly suggested that the blood vessels are deeply involved in the excretion and volume regulation of the tissue fluid.

Structure of the rat subcutaneous connective tissue in relation to its sliding mechanism.

Mammalian skin can extensively slide over most parts of the body. To study the mechanism of this mobility of the skin, the structure of the subcutaneous connective tissue was examined by light microscopy. The subcutaneous connective tissue was observed to be composed of multiple layers of thin collagen sheets containing elastic fibers. These piled-up collagen sheets were loosely interconnected with each other, while the outer and inner sheets were respectively anchored to the dermis and epimysium by elastic fibers. Collagen fibers in each sheet were variable in diameter and oriented in different directions to form a thin, loose meshwork under conditions without mechanical stretching. When a weak shear force was loaded between the skin and the underlying abdominal muscles, each collagen sheet slid considerably, resulting in a stretching of the elastic fibers which anchor these sheets. When a further shear force was loaded, collagen fibers in each sheet seemed to align in a more parallel manner to the direction of the tension. With the reduction or removal of the force, the arrangement of collagen fibers in each sheet was reversed and the collagen sheets returned to their original shapes and positions, probably with the stabilizing effect of elastic fibers. Blood vessels and nerves in the subcutaneous connective tissue ran in tortuous routes in planes parallel to the unloaded skin, which seemed very adaptable for the movement of collagen sheets. These findings indicate that the subcutaneous connective tissue is extensively mobile due to the presence of multilayered collagen sheets which are maintained by elastic fibers.