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.

Effect of intravitreal bevacizumab on iris vessels in neovascular glaucoma patients.

BACKGROUND: We aimed to investigate the effects of a single 1-mg injection of intravitreal bevacizumab on iris vessels in neovascular glaucoma (NVG) patients. METHODS: Twenty-two surgically resected irises from glaucoma patients were obtained during trabeculectomy. Eight were from patients with NVG who received a 1-mg injection of intravitreal bevacizumab (IVB) before glaucoma surgery, eight were from patients with primary open-angle glaucoma (POAG), and six were from patients with NVG who were not administered IVB. The collected iris specimens were compared after immunohistochemical staining with anti-CD34 monoclonal antibodies and anti-VEGF monoclonal antibody, and the percentage of CD34-positive and VEGF-positive regions in the total area of the specimens from the three groups was compared. RESULTS: The difference in the CD34-positive area between all groups was statistically significant (p = 0.0061, Kruskal-Wallis test). There was no significant difference in the CD34-positive area between the NVG with IVB group and the POAG group (p = 0.3017, Mann-Whitney U test with Bonferroni correction). The POAG group had significantly fewer CD34-positive regions than the NVG without IVB group (p = 0.0019, Mann-Whitney U test with Bonferroni correction). Many vessels remained in the iris stroma, and there was no significant difference in the CD34-positive area between the NVG with IVB and NVG without IVB groups (p = 0.0357, Mann-Whitney U test with Bonferroni correction). The ratio of the length of CD34 expression on the iris surface in the NVG without IVB group was significantly longer than that in the NVG with IVB group (p = 0.0002, Mann-Whitney U test). The difference in VEGF expression between all groups was statistically significant (p = 0.04, Kruskal-Wallis test). There was no significant difference between the NVG with IVB group and the NVG without IVB group (p = 0.7963 Mann-Whitney U test with Bonferroni correction). The frequency of hyphema and fibrin formation in the anterior chamber 1 day after surgery between the two NVG groups was not statistically significant. CONCLUSION: A single intravitreal dose of IVB at 1 mg/0.04 ml to eyes with rubeotic glaucoma reduced the neovascularization in the human iris surface, but could not eliminate completely neovascularization in iris stroma. This finding implies that the prevention of hyphema and fibrin formation based on the slit-lamp examination can not be predicted, even if neovascularization in iris surface seems to be eliminated by a single dose of IVB.

Regeneration of peripheral nerve after transplantation of CD133+ cells derived from human peripheral blood.

OBJECT: Despite intensive efforts in the field of peripheral nerve injury and regeneration, it remains difficult to achieve full functional recovery in humans following extended peripheral nerve lesions. In this study, the authors examined the use of blood-derived CD133(+) cells in promoting the repair of peripheral nerve defects. METHODS: The authors transplanted phosphate-buffered saline (control), mononuclear cells, or CD133(+) cells embedded in atelocollagen gel into a silicone tube that was used to bridge a 15-mm defect in the sciatic nerve of athymic rats (12 animals in each group). At 8 weeks postsurgery, molecular, histological, and functional evaluations were performed in regenerated tissues. RESULTS: The authors found that sciatic nerves in which a defect had been made were structurally and functionally regenerated within 8 weeks after CD133(+) cell transplantation. From macroscopic evaluation, massive nervelike tissues were confirmed only in rats with CD133(+) cell transplantation compared with the other groups. Morphological regeneration in the samples after CD133(+) cell transplantation, as assessed using toluidine blue staining, was enhanced significantly in terms of the number of myelinated fibers, axon diameter, myelin thickness, and percentage of neural tissue. Compound muscle action potentials were observed only in CD133(+) cell-treated rats. Furthermore, it was demonstrated that the transplanted CD133(+) cells differentiated into Schwann cells by 8 weeks after transplantation. CONCLUSIONS: The results show that CD133(+) cells have potential for enhancement of histological and functional recovery from peripheral nerve injury. This attractive cell source could be purified easily from peripheral blood and could be a feasible autologous candidate for peripheral nerve injuries in the clinical setting.

Role of osteoprotegerin in arterial calcification: development of new animal model.

OBJECTIVES: Enhanced osteoclastogenesis, increased bone resorption, and osteoporosis have been reported in osteoprotegerin-deficient (OPG (-/-)) mice. OPG (-/-) mice available in Japan usually do not show vascular calcification. We have found that arterial calcification can be quickly induced by a simple procedure in OPG (-/-) mice. METHODS AND RESULTS: Male OPG (-/-), OPG (+/-), and OPG (+/+) mice were fed a high phosphate diet from 6 to 10 weeks after birth, and then 1alpha,25-dihydroxyvitamin D3 (calcitriol) was injected for 3 days. We found that severe calcification developed in the media of the aorta in OPG (-/-) mice. Under electron microscopy, calcium deposits were observed in the cytoplasm and extracellular matrix of vascular smooth muscle cells (VSMCs). Neither apoptosis of VSMCs nor infiltration of macrophages was observed. Alkaline phosphatase (ALP) activity of aortic tissue correlated with the calcified lesion area. Mouse aorta and bone extracts revealed an identical pattern by ALP electrophoresis. CONCLUSIONS: Our results demonstrated that OPG had anticalcification activity in the aorta, probably through the downregulation of ALP activity. Because the time course of arterial calcification after the injection of calcitriol is accurate and reproducible, this mouse model will be useful for further investigation of vascular calcification.

Development of arthrogenic joint contracture as a result of pathological changes in remobilized rat knees.

This study aimed to elucidate how rats recover from immobilization-induced knee joint contracture. Rats' right knees were immobilized by an external fixator at a flexion of 140° for 3 weeks. After removal of the fixator, the joints were allowed to move freely (remobilization) for 0, 1, 3, 7, or 14 days (n = 5 each). To distinguish myogenic and arthrogenic contractures, the passive extension range of motion was measured before and after myotomy of the knee flexors. Knee joints were histologically analyzed and the expression of genes encoding inflammatory or fibrosis-related mediators, interleukin-1ß (1L-1ß), fibrosis-related transforming growth factor-ß1 (TGF-ß1), and collagen type I (COL1A1) and III (COL3A1), were examined in the knee joint posterior capsules using real-time PCR. Both myogenic and arthrogenic contractures were established within 3 weeks of immobilization. During remobilization, the myogenic contracture decreased over time. In contrast, the arthrogenic contracture developed further during the remobilization period. On day 1 of remobilization, inflammatory changes characterized by edema, inflammatory cell infiltration, and upregulation of IL-1ß gene started in the knee joint posterior capsule. In addition, collagen deposition accompanied by fibroblast proliferation, with upregulation of TGF-ß1, COL1A1, and COL3A1 genes, appeared in the joint capsule between days 7 and 14. These results suggest the progression of arthrogenic contracture following remobilization, which is characterized by fibrosis development, is possibly triggered by inflammation in the joint capsule. It is therefore necessary to focus on developing new treatment strategies for immobilization-induced joint contracture. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1414-1423, 2017.

Effects of CD44 antibody-- or RGDS peptide--immobilized magnetic beads on cell proliferation and chondrogenesis of mesenchymal stem cells.

We evaluated the efficacy of a novel mesenchymal stem cell (MSC) delivery system using an external magnetic field for cartilage repair in vitro. MSCs were isolated from the bone marrow of Sprague Drawley rats and expanded in a monolayer. To use the MSC delivery system, two types of MSC-magnetic bead complexes were designed and compared. Expanded MSCs were combined with small-sized (diameter: 310 nm) carboxyl group-combined (0.01-0.04 micromol/mg) magnetic beads, Ferri Sphere 100C, through either anti-rat CD44 mouse monoclonal antibodies or a synthetic cell adhesion factor, arginine (R)-glycine (G)-aspartic acid (D)-serine (S) (RGDS) peptide. Both cell complexes were successfully created, and were able to proliferate in monolayer culture up to at least day 7 after separation of magnetic beads from the cell surface, although the proliferation of the complexes was slower in the early period of culture than that of non-labeled rat MSCs (after 7 days of culture: proliferation of CD44 antibody-bead complexes, approximately 50%; RGDS peptide-bead complexes, 70% versus non-labeled rat MSCs, respectively). These complexes were seeded onto culture plates with or without an external magnetic force (magnetic flux density was 0.20 Tesla at a distance of 2 mm from plate base) generated by a neodymium magnet, and supplemented with chondrogenic differentiation medium. Both complexes could be attached and gathered effectively under the influence of the external magnet, and CD44-bead complexes could effectively generate chondrogenic matrix in monolayer culture. In a three-dimensional culture system, the production of a dense chondrogenic matrix and the expression of type II collagen and aggrecan mRNA were detected in both complexes, and the chondrogenic potential of these complexes was only a little less than that of rat MSCs alone. Thus, we conclude that due to the fact that MSC-RGDS peptide-bead complexes are composed using a biodegradable material, RGDS peptide, as a mediator, the RGDS peptide-bead complex is more useful for minimally invasive clinical applications using our design of magnetic MSC delivery system than CD44 antibody-beads.

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.

Characterization of labeled neural progenitor cells for magnetic targeting.

For many diseases and injuries of the central nervous system, transplantation of neural progenitor cells is being evaluated as a possible treatment option. Although local, intravenous and subarachnoid injections have been reported as administration methods of neural progenitor cells, each of these methods has limitations. More effective and minimally invasive cell delivery systems are necessary for transplanting neural progenitor cells. In this study, we have developed a technique to form magnetically labeled neural progenitor cells for a magnetic targeting system. We demonstrated that neural progenitor cells can couple with magnetic beads, and that the labeled neural progenitor cells preserve the characteristics of non-labeled neural progenitor cells, and that they can be localized by magnetic force in vitro. Labeled neural progenitor cells have the potential to be used in magnetic targeting systems in-vivo models.

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.

Localization and hormonal control of serine dehydratase during metabolic acidosis differ markedly from those of phosphoenolpyruvate carboxykinase in rat kidney.

Serine dehydratase (SDH) is abundant in the rat liver but scarce in the kidney. When administrated with dexamethasone, the renal SDH activity was augmented 20-fold, whereas the hepatic SDH activity was affected little. In situ hybridization and immunohistochemistry revealed that SDH was localized to the proximal straight tubule of the nephron. To address the role of this hormone, rats were made acidotic by gavage of NH(4)Cl. Twenty-two hours later, the SDH activity was increased three-fold along with a six-fold increment in the phosphoenolpyruvate carboxykinase (PEPCK) activity, a rate-limiting enzyme of gluconeogenesis. PEPCK, which is localized to the proximal tubules under the normal condition, spreads throughout the entire cortex to the outer medullary rays by acidosis, whereas SDH does not change regardless of treatment with dexamethasone or NH(4)Cl. When NH(4)Cl was given to adrenalectomized rats, in contrast to the SDH activity no longer increasing, the PEPCK activity responded to acidosis to the same extent as in the intact rats. A simultaneous administration of dexamethasone and NH(4)Cl into the adrenalectomized rats fully restored the SDH activity, demonstrating that the rise in the SDH activity during acidosis is primarily controlled by glucocorticoids. The present findings clearly indicate that the localization of SDH and its hormonal regulation during acidosis are strikingly different from those of PEPCK.

Reduction of formaldehyde concentrations in the air and cadaveric tissues by ammonium carbonate.

The reduction of formaldehyde by ammonium carbonate was examined in cadavers and in vitro. Formaldehyde concentrations in the air (10 cm above human cadavers) and in various cadaveric tissues were measured with or without perfusion of ammonium carbonate solution into formaldehyde-fixed cadavers. Air samples were monitored using Kitagawa gas detector tubes. For measurement of formaldehyde in tissues, muscles and organs were cut into small pieces and tissue fluids were separated out by centrifugation. These specimen fluids were diluted, supplemented with 3-methyl-2-benzothiazolinone hydrazone hydrochloride and quantified by spectrophotometry. In five cadavers without ammonium carbonate treatment, the formaldehyde concentrations in the air above the thorax and in various tissue fluids were 1.2-3.0 p.p.m. and 0.15-0.53%, respectively. Arterial reperfusion of saturated ammonium carbonate solution (1.0, 1.5 or 2.0 L) into five formaldehyde-fixed cadavers successfully reduced the formaldehyde levels, both in the air (0.5-1.0 p.p.m.) and in various tissue fluids (0.012-0.36%). In vitro experiments demonstrated that formaldehyde concentrations decreased, first rapidly and then gradually, with the addition of ammonium carbonate solution into fluids containing formaldehyde. It was confirmed that formaldehyde reacted with the ammonium carbonate and was thereby changed into harmless hexamethylenetetramine. The application of ammonium carbonate solution via intravascular perfusion and, if necessary, by infusion into the thoracic and peritoneal cavities, injection into muscles and spraying on denuded tissues can be anticipated to reduce formaldehyde to satisfactorily low levels in cadaveric tissues and, consequently, in the air, which may provide safe and odorless dissecting rooms.

Comparison of sarcomere alterations after muscle contraction and tension loading in the rat soleus muscle.

Muscle contraction induced by 30 min of continuous nerve stimulation at 50 Hz resulted in sarcomere changes of the soleus muscle in the rat in our previous study. To further investigate the cause of sarcomere alterations, the sciatic nerve was electrically stimulated intermittently for 30 min. Nerve stimulation was also conducted after cutting the tendons of the soleus, gastrocnemius and plantaris muscles in order to prevent imposing tension on these muscles as a result to their own contractions. In addition, the muscles were pulled by weights via their tendons to load high tension for 30 min without nerve stimulation. Sarcomere alterations immediately after treatments were quantified by electron microscopy. The percentages of aberrant sarcomere areas of the soleus muscle were 25.7 +/- 16.4% (mean +/- SD) in the group of intermittent nerve stimulation with intact tendons and 21.1 +/- 35.4% in the group of tenotomy and continuous nerve stimulation, which were roughly equal to or more severe than the group of continuous nerve stimulation with intact tendons (18.8 +/- 15.8%) in our previous study. Sarcomere alterations consisted mainly of hypercontraction in these groups. Almost all sarcomere changes in the tension-loaded (pulled) soleus muscles were scarce myofilaments (1.7 +/- 1.0% by 600 g; 4.5 +/- 2.9% by 1200 g), and hypercontraction was not observed. These findings indicate that neither high tension nor a decrease of muscle blood flow during continuous contraction seems to be the primary cause of sarcomere alterations in the present study. There are probably other causes that produce aberrant sarcomeres.

Fiber dissection technique for demonstrating the lateral lemniscus of the human brain.

This report describes fiber dissection technique for tracing the auditory pathway from the cochlear nerve to the medial geniculate body via the lateral lemniscus, inferior colliculus and inferior brachium. Some fibers of the lateral leminiscus appear to reach the thalamus in conjunction with fibers of the medial lemniscus.

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.

Histological findings of uveal capillaries in rabbit eyes after multiple intravitreal injections of bevacizumab.

PURPOSE: To evaluate the potential toxicity of multiple intravitreal injections of bevacizumab on the uveal capillaries of rabbit eyes. MATERIALS AND METHODS: Nine eyes of nine rabbits that received single intravitreal injections of bevacizumab (IVB) constituted the single IVB group, while nine eyes of nine rabbits that received three injections of IVB, with an interval of 28 days between injections, constituted the repeat IVB group. Seven eyes of seven rabbits constituted the control group. The rabbits in the single and repeat IVB groups were sacrificed 7 and 28 d after the single and third IVB injection, respectively. Uveal specimens were compared between groups after immunohistochemical staining. Ultrastructural findings were evaluated by electron microscopy. Control group rabbits were sacrificed 7 d after saline injection. Clinical examination and fundus fluorescein angiography were performed at baseline, 7 d after the first injection, and after the last injection. RESULTS: Differences in the CD31-positive areas of the iris, ciliary body and choroid 7 d after IVB were not statistically significant among the single IVB, repeat IVB and control groups (p = 0.0749, p = 0.7237 and p = 0.7346, respectively; analysis of variance). Endothelial cell fenestrations (ECFs) in the choriocapillaris and ciliary body observed by electron microscopy on day 7 in the single and repeat IVB groups were decreased by 50% (p < 0.0001) and 33% (p < 0.0001), respectively, in both IVB groups compared with those in the control group. However, ECFs observed on day 28 in both groups were comparable to those observed in the control group. CONCLUSIONS: Single IVB and repeated IVB did not have any effect on normal vessel endothelium density as per immunohistochemical findings. Ultrastructural findings revealed that IVB transiently decreased the ECFs in the choriocapillaris and ciliary body.

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.