Year-long upregulation of connexin43 in rabbit hearts by heavy ion irradiation.

A previous study from our laboratory has shown that a single targeted heavy ion irradiation (THIR; 15 Gy) to rabbit hearts increases connexin43 (Cx43) expression for 2 wk in association with an improvement of conduction, a decrease of the spatial inhomogeneity of repolarization, and a reduction of vulnerability to ventricular arrhythmias after myocardial infarction. This study investigated the time- and dose-dependent effects of THIR (5-15 Gy) on Cx43 expression in normal rabbit hearts (n = 45). Five rabbits without THIR were used as controls. A significant upregulation of Cx43 protein and mRNA in the ventricular myocardium was recognized by immunohistochemistry, Western blotting, and real-time PCR from 2 wk up to 1 yr after a single THIR at 15 Gy. THIR > or =10 Gy caused a significant dose-dependent increase of Cx43 protein and mRNA 2 wk after THIR. Anterior, lateral, and posterior free wall of the left ventricle, interventricular septum, and right ventricular free wall were affected similarly by THIR in terms of Cx43 upregulation. The radiation-induced increase of immunolabeled Cx43 was observed not only at the intercalated disk region but also at the lateral surface of ventricular myocytes. The increase of immunoreactive Cx43 protein was predominant in the membrane fraction insoluble in Triton X-100, that is the Cx43 in the sarcolemma. In vivo examinations of the rabbits 1 yr after THIR (15 Gy) revealed no significant changes in ECGs and echocardiograms (left ventricular dimensions, contractility, and diastolic function), indicating no apparent late radiation injury. A single application of THIR causes upregulation and altered cellular distribution of Cx43 in the ventricles lasting for at least 1 yr. This long-lasting remodeling effect on gap junctions may open the pathway to novel therapy against life threatening ventricular arrhythmias in structural heart disease.

Validation of the Friedewald Equation for Evaluation of Plasma LDL-Cholesterol.

In most clinical laboratories, low density lipoprotein (LDL) cholesterol is usually estimated indirectly with the Friedewald equation or directly with the N-geneous assay. We assessed LDL-cholesterol values obtained by both methods to find an appropriate fasting period and to assess the influence of the energy content of the last meal. Blood samples were taken from 28 healthy volunteers who had consumed a standard meal (107 g of carbohydrate, 658 kcal) followed by a fasting period of 12 and 18 h, or a high-energy meal (190 g of carbohydrate, 1011 kcal) with a fasting period of 12 h. Prolongation of the fasting period from 12 h to 18 h decreased glucose level, but did not decrease triacylglycerol, total cholesterol, or high density lipoprotein (HDL) cholesterol. LDL-cholesterol levels measured with the N-geneous assay did not change (94.0 +/- 21.5 to 96.3 +/- 19.1 mg/dl). LDL-cholesterol levels calculated with the Friedewald equation were also similar after fasting periods of 12 h (98.5 +/- 21.4 mg/dl) and 18 h (99.7 +/- 20.2 mg/dl). The high-energy meal did not change the level of LDL-cholesterol measured with the N-geneous assay (96.1 +/- 21.2 mg/dl), or the glucose, triacylglycerol, total cholesterol, or HDL-cholesterol level, but LDL-cholesterol levels evaluated from the Friedewald equation (92.6 +/- 20.3 mg/dl) became significantly lower. A fasting time longer than 12 h is not necessary to obtain reasonable blood lipid levels. The Friedewald equation gave higher LDL-cholesterol levels than N-geneous assay in young Japanese females who had eaten a low-energy meal, and lower values when they had eaten a high-energy meal. Thus, it may be necessary to pay attention to energy of nigh meal prior to blood withdrawal.

Heavy ion radiation up-regulates Cx43 and ameliorates arrhythmogenic substrates in hearts after myocardial infarction.

OBJECTIVE: Radiation has been shown to enhance intercellular communication in the skin and lungs through an increase of connexin43 (Cx43) expression. If analogous Cx43 up-regulation is induced in the diseased heart, it would provide a new perspective in radiation therapy for arrhythmias. The aim of the present study is to test this hypothesis. METHODS: Non-transmural myocardial infarction (MI) was created in 24 rabbits by microsphere injection into the coronary arteries. Twenty-four rabbits without MI were used as controls. Targeted external heavy ion beam irradiation (THIR; 15 Gy) was applied 2 weeks after MI with an accelerator (HIMAC, Chiba, Japan). RESULTS: The THIR was associated with an increase of Cx43 mRNA and protein levels in the left ventricle in control as well as in MI rabbits. THIR also increased lateralization of Cx43, which was no longer colocalized with cadherins. In MI hearts, immunoreactive Cx43 signals were reduced in the peri-infarct zone, and the reduction was reversed by THIR. In-vivo epicardial potential mapping on the free wall (64 unipolar electrodes to cover 7 x 7 mm) in MI hearts revealed reduced conduction velocity, whereas dispersion of the activation-recovery interval (ARI) was increased compared with controls, and these changes were reversed by THIR. The vulnerability for ventricular tachyarrhythmias (VT/VF), which was estimated by programmed stimulation, was increased in MI hearts, and this increased vulnerability to arrhythmias was reversed by THIR. CONCLUSIONS: THIR increases Cx43 expression, improves the conductivity, decreases the spatial heterogeneity of repolarization, and reduces the vulnerability of rabbit hearts to ventricular arrhythmias after MI. THIR could have an antiarrhythmic potential through an improvement of electrical coupling.

Beneficial effect of hydroxyfasudil, a specific Rho-kinase inhibitor, on ischemia/reperfusion injury in canine coronary microcirculation in vivo.

OBJECTIVES: We examined whether hydroxyfasudil, a specific Rho-kinase inhibitor, exerts cardioprotective effect on coronary ischemia/reperfusion (I/R) injury and, if so, whether nitric oxide (NO) is involved. BACKGROUND: Recent studies have demonstrated that Rho-kinase is substantially involved in the pathogenesis of cardiovascular diseases; however, it remains to be examined whether it is also involved in ischemia/reperfusion (I/R) injury. METHODS: Canine subepicardial small arteries (SA, >or=100 microm) and arterioles (A, <100 microm) were observed by a charge-coupled device intravital microscope during I/R. Coronary vascular responses to endothelium-dependent (acetylcholine, intracoronary [IC]) and -independent (papaverine, IC) vasodilators were examined after I/R under the following four conditions: control (n = 7), NO synthase inhibitor alone (N(G)-monomethl-L-arginine [L-NMMA], IC, n = 4), hydroxyfasudil alone (IC, n = 7), and hydroxyfasudil plus L-NMMA (n = 7). RESULTS: Hydroxyfasudil significantly attenuated serotonin (IC)-induced vasoconstriction of SA (-7 +/- 1% vs. 2 +/- 1%, p < 0.01). Coronary I/R significantly impaired coronary vasodilation to acetylcholine after I/R (SA, p < 0.05; and A, p < 0.01 vs. before I/R) and L-NMMA further reduced the vasodilation, whereas hydroxyfasudil completely preserved the responses. The vasoconstriction by L-NMMA after I/R was significantly improved by hydroxyfasudil in both-sized arteries (both p < 0.01). Expression of endothelial nitric oxide synthase (eNOS) protein in the ischemic endocardium of left anterior descending coronary artery area (as determined by Western blotting) significantly decreased (79 +/- 4%) compared with the nonischemic endocardium of LCX area (100 +/- 7%), which was improved by hydroxyfasudil (105 +/- 6%, p < 0.01). Hydroxyfasudil significantly reduced myocardial infarct size, and hydroxyfasudil with L-NMMA also reduced the infarct size compared with L-NMMA alone. CONCLUSIONS: Hydroxyfasudil exerts cardioprotective effects on coronary I/R injury in vivo, in which NO-mediated mechanism may be involved through preservation of eNOS expression.

Treatment of natto, a fermented soybean preparation, to prevent excessive plasma vitamin K concentrations in patients taking warfarin.

The purpose of this study is to find a method of cooking natto that prevents the appearance of high-plasma vitamin K concentrations after the consumption of natto, so that patients taking warfarin can benefit from eating natto. Five cooking methods were examined to determine which could most effectively decrease the count of the living Bacillus subtilis in natto. Volunteers ate natto or treated natto, and their plasma vitamin K level was measured at 5, 8, 24 and 48 h thereafter. One gram of natto contained 9.7+/-0.1 Log cfu/mL of Bacillus subtilis. Boiling significantly reduced the Bacillus subtilis count to 5.1+/-0.3 Log cfu/mL, and concomitantly reduced the content of menaquinone-7 (MK-7), which is a form of vitamin K synthesized by Bacillus subtilis, from 660.40+/-65.32 ng/mL to 78.50+/- 11.12 ng/mL. Untreated natto increased the MK-7 concentration in blood from 1.86+/-1.51 ng/mL to 14.54+/-4.12 ng/mL at 5 h after intake, and the MK-7 concentration remained elevated at 8, 24 and 48 h (7.29+/-2.20, 6.97+/-2.60, and 5.37+/-1.94 ng/mL, respectively). In contrast, boiled natto increased plasma MK-7 only mildly (from 1.61+/-1.11 to 4.02+/-0.82 ng/ mL at 5 h) and the concentration remained relatively stable up to 48 h (3.46+/-0.83, 4.22+/-1.51 and 2.77+/-0.75 ng/mL at 8, 24 and 48 h, respectively). In conclusion, boiled natto did not cause a marked increase in the plasma concentration of vitamin K in subjects who consumed it. Thus, patients on warfarin may be able to eat boiled natto without ill effects.

Adrenomedullin gene transfer induces therapeutic angiogenesis in a rabbit model of chronic hind limb ischemia: benefits of a novel nonviral vector, gelatin.

BACKGROUND: Earlier studies have shown that adrenomedullin (AM), a potent vasodilator peptide, has a variety of cardiovascular effects. However, whether AM has angiogenic potential remains unknown. This study investigated whether AM gene transfer induces therapeutic angiogenesis in chronic hind limb ischemia. METHODS AND RESULTS: Ischemia was induced in the hind limb of 21 Japanese White rabbits. Positively charged biodegradable gelatin was used to produce ionically linked DNA-gelatin complexes that could delay DNA degradation. Human AM DNA (naked AM group), AM DNA-gelatin complex (AM-gelatin group), or gelatin alone (control group) was injected into the ischemic thigh muscles. Four weeks after gene transfer, significant improvements in collateral formation and hind limb perfusion were observed in the naked AM group and AM-gelatin group compared with the control group (calf blood pressure ratio: 0.60+/-0.02, 0.72+/-0.03, 0.42+/-0.06, respectively). Interestingly, hind limb perfusion and capillary density of ischemic muscles were highest in the AM-gelatin group, which revealed the highest content of AM in the muscles among the three groups. As a result, necrosis of lower hind limb and thigh muscles was minimal in the AM-gelatin group. CONCLUSIONS: AM gene transfer induced therapeutic angiogenesis in a rabbit model of chronic hind limb ischemia. Furthermore, the use of biodegradable gelatin as a nonviral vector augmented AM expression and thereby enhanced the therapeutic effects of AM gene transfer. Thus, gelatin-mediated AM gene transfer may be a new therapeutic strategy for the treatment of peripheral vascular diseases.

Biodegradable gelatin hydrogel potentiates the angiogenic effect of fibroblast growth factor 4 plasmid in rabbit hindlimb ischemia.

OBJECTIVES: We investigated the potentiation of gene therapy using fibroblast growth factor 4 (FGF4)-gene by combining plasmid deoxyribonucleic acid (DNA) with biodegradable gelatin hydrogel (GHG). BACKGROUND: Virus vectors transfer genes efficiently but are biohazardous, whereas naked DNA is safer but less efficient. Deoxyribonucleic acid charges negatively; GHG has a positively charged structure and is biodegradable and implantable; FGF4 has an angiogenic ability. METHODS: The GHG-DNA complex was injected into the hindlimb muscle (63 mice and 55 rabbits). Gene degradation was evaluated by using (125)I-labeled GHG-DNA complex in mice. Transfection efficiency was evaluated with reverse-transcription nested polymerase chain reaction and X-Gal histostaining. The therapeutic effects of GHG-FGF4-gene complex (GHG-FGF4) were evaluated in rabbits with hindlimb ischemia. RESULTS: Gelatin hydrogel maintained plasmid in its structure, extending gene degradation temporally until 28 days after intramuscular delivery, and improving transfection efficiency. Four weeks after gene transfer, hindlimb muscle necrosis was ameliorated more markedly in the GHG-FGF4 group than in the naked FGF4-gene and GHG-beta-galactosidase (control) groups (p < 0.05, Kruskal-Wallis test). Synchrotron radiation microangiography (spatial resolution, 20 microm) and flow determination with microspheres confirmed significant vascular responsiveness to adenosine administration in the GHG-FGF4 group, but not in the naked FGF4-gene and the control. CONCLUSIONS: The GHG-FGF4 complex promoted angiogenesis and blood flow regulation of the newly developed vessels possibly by extending gene degradation and improving transfection efficiency without the biohazard associated with viral vectors.

Compact monochromatic flash x-ray generator utilizing a disk-cathode molybdenum tube.

The high-voltage condensers in a polarity-inversion two-stage Marx surge generator are charged from -50 to -70 kV by a power supply, and the electric charges in the condensers are discharged to an x-ray tube after closing gap switches in the surge generator with a trigger device. The x-ray tube is a demountable diode, and the turbo molecular pump evacuates air from the tube with a pressure of approximately 1 mPa. Clean molybdenum Kalpha lines are produced using a 20 microm-thick zirconium filter, since the tube utilizes a disk cathode and a rod target, and bremsstrahlung rays are not emitted in the opposite direction to that of electron acceleration. At a charging voltage of -70 kV, the instantaneous tube voltage and current were 120 kV and 1.0 kA, respectively. The x-ray pulse widths were approximately 70 ns, and the generator produced instantaneous number of Kalpha photons was approximately 3 x 10(7) photons/cm2 per pulse at 0.5 m from the source of 3.0 mm in diameter.

Severity of white matter hyperintensities in older adults aged in their mid 80s and its relationship with hemodynamic and cardiac parameters.

AIM: In the present study, we analyzed the severity of white matter hyperintensities detected on magnetic resonance imaging of the brain in older Japanese adults who were aged in their mid 80s, and examined its relationships with the clinical parameters. METHODS: To identify factors related to the severity of white matter hyperintensities in 33 older adult attendees of our outpatient clinic and 17 older adults living in a group home, we carried out logistic regression analyses and/or correlation analyses. RESULTS: Cognitive function and activities of daily living were significantly correlated with the severity of white matter hyperintensities. Multivariate analysis identified activities of daily living, but not cognitive function, as being independently associated with the severity of white matter hyperintensities. Several hemodynamic and cardiac parameters, including diastolic blood pressure, hemoglobin, serum level of N-terminal pro-brain natriuretic peptide, cardiothoracic ratio on the chest X-ray, severity of supraventricular arrhythmias on a Holter electrocardiogram and serum levels of docosahexaenoic acid, were significantly correlated with the severity of white matter hyperintensities. In contrast, the serum cholesterol, glycosylated hemoglobin value and systolic blood pressure were not correlated with the severity of white matter hyperintensities. CONCLUSIONS: The severity of white matter hyperintensities detected on magnetic resonance imaging of the brain in older Japanese patients aged in their mid 80s was significantly correlated with activities of daily living, hemodynamic and cardiac parameters, and the serum level of docosahexaenoic acid.

Altered blood flow in cerebral perforating arteries of rat models of diabetes: A synchrotron radiation microangiographic study toward clinical evaluation of white matter hyperintensities.

AIM: As altered blood flow in the cerebral perforating arteries (PA) might be related to development of cerebral white matter hyperintensities, we examined whether the hemodynamic relationship of the PA and middle cerebral artery (MCA) is altered in rat models of diabetes, compared with normal rats and a rat model of sinoatrial denervation (blood pressure fluctuation model). METHODS: We used microangiography with monochromatic synchrotron radiation to measure the diameters of the PA and MCA at 4.5 µm resolution in five groups of rats: (i) Long-Evans Tokushima Otsuka (LETO); (ii) Otsuka Long-Evans Tokushima Fatty (a model of type 2 diabetes with obesity); (iii) LETO with sinoaortic denervation (LETO + SAD); (iv) F344; and (v) F344 + streptozotocin (a model of type 1 diabetes). RESULTS: Compared with LETO, Otsuka Long-Evans Tokushima Fatty rats showed a significant reduction in the diameter of both PA and MCA, though the PA/MCA diameter ratio was unchanged. In contrast, compared with LETO, LETO + SAD rats showed an increased MCA diameter, and the PA/MCA diameter ratio was decreased. Compared with F344 rats, the MCA diameter was increased in F344 + streptozotocin rats, and the PA/MCA diameter ratio was decreased. Scatter diagrams showed that the diameters of the PA and MCA were essentially independent of each other in the two types of diabetic models. CONCLUSION: PA were consistently visualized at high resolution by means of microangiography using synchrotron radiation. The present results show that rat diabetic models exhibit changes in PA diameter and PA/MCA diameter ratio, which might be related to the development of diabetes-associated cerebral white matter hyperintensities.

Demonstration of enhanced K-edge angiography using a cerium target x-ray generator.

The cerium target x-ray generator is useful in order to perform enhanced K-edge angiography using a cone beam because K-series characteristic x rays from the cerium target are absorbed effectively by iodine-based contrast mediums. The x-ray generator consists of a main controller, a unit with a Cockcroft-Walton circuit and a fixed anode x-ray tube, and a personal computer. The tube is a glass-enclosed diode with a cerium target and a 0.5-mm-thick beryllium window. The maximum tube voltage and current were 65 kV and 0.4 mA, respectively, and the focal-spot sizes were 1.0 x 1.3 mm. Cerium Kalpha lines were left using a barium sulfate filter, and the x-ray intensity was 0.48 microC/kg at 1.0 m from the source with a tube voltage of 60 kV, a current of 0.40 mA, and an exposure time of 1.0 s. Angiography was performed with a computed radiography system using iodine-based microspheres. In coronary angiography of nonliving animals, we observed fine blood vessels of approximately 100 microm with high contrasts.

Inhomogeneous vasodilatory responses of rat tail arteries to heat stress: evaluation by synchrotron radiation microangiography.

Tail blood flow is crucial for dissipating body heat in rats. Angiographies are convenient tools to evaluate tail circulation. However, conventional angiographies do not have sufficient sensitivity or spatial resolution for small vessels. Recently, we developed a novel microangiographic system using monochromatic synchrotron radiation and a high-definition video camera system. Here, we report an evaluation of rat tail circulation under heat stress using the synchrotron radiation microangiographic system. We performed an experiment using the microangiography of the caudal artery before and after heating up WKAH/HkmSlc rats to rectal temperature of 39 degrees C. The images were digitized and temporal subtraction was performed, and the diameters of caudal arteries were evaluated. After heating, the medial caudal artery was markedly dilated (320 +/- 53 to 853 +/- 243 micro m in diameter, p<0.001), while no significant change was observed in the lateral caudal arteries (139 +/- 42 to 167 +/- 73 micro m) and segmental anastomosing vessels. The heat stress allowed for visualization of the superficial caudal arteries with a diameter of approximately 60 micro m, not visible prior to heating. Thus, synchrotron radiation microangiography demonstrated that the rat tail possessed dual sets of arteries; one set was highly sensitive to heat-induced vasodilation (medial caudal artery and superficial caudal arteries) and the other set was less sensitive (lateral caudal arteries and segmental anastomosing vessels).

Induction of vascular endothelial growth factor by fibrin as a dermal substrate for cultured skin substitute.

In the initial phase of wound healing, endogenous fibrin clots are known to form a provisional matrix and to promote angiogenesis. Growth factors such as vascular endothelial growth factor (VEGF) increase in wounds to stimulate angiogenesis. However, it remains unknown whether VEGF is induced when fibrin is used as a dermal substrate for cultured skin substitutes. The authors investigated the effect of fibrin gel as a dermal substrate for a cultured skin substitute, using human keratinocytes and dermal fibroblasts. A collagen-cultured skin substitute was also examined for comparison. VEGF in the culture supernatant in both types was measured by enzyme-linked immunosorbent assay, and VEGF mRNA was determined semiquantitatively by reverse-transcriptase polymerase chain reaction after 2 days of incubation. Experiments were performed using 12 cultured skin substitutes: four for histologic examination before transplantation, four for VEGF assay in vitro, and four for the transplantation to athymic mice. Three independent experiments were performed for each step. VEGF concentration in the fibrin-cultured supernatant was 84.3 +/- 11.8 pg/ml, whereas it was 27.8 +/- 4.68 pg/ml in the case of the collagen substrate. The relative levels of VEGF mRNA were 1.088 +/- 0.100 and 0.698 +/- 0.226, respectively. In in vivo transplantation, the fibrin-type cultured skin substitute showed an excellent take on the wound bed, and a normally proliferating keratinocyte layer with emergence of vascular endothelial cells in the transplanted floor was seen 3 days after transplantation. Vascular endothelial cells, which were identified using alkaline phosphatase stain, were significantly increased in the fibrin-type cultured skin substitute. The use of fibrin as a dermal substrate for cultured skin substitute increases the secretion of VEGF, improves regeneration of mature epidermal structure after in vivo transplantation, and promotes the migration of vascular endothelial cells.

Demonstration of iodine K-edge imaging by use of an energy-discrimination X-ray computed tomography system with a cadmium telluride detector.

An energy-discrimination K-edge X-ray computed tomography (CT) system is useful for increasing the contrast resolution of a target region by utilizing contrast media. The CT system has a cadmium telluride (CdTe) detector, and a projection curve is obtained by linear scanning with use of the CdTe detector in conjunction with an X-stage. An object is rotated by a rotation step angle with use of a turntable between the linear scans. Thus, CT is carried out by repetition of the linear scanning and the rotation of an object. Penetrating X-ray photons from the object are detected by the CdTe detector, and event signals of X-ray photons are produced with use of charge-sensitive and shaping amplifiers. Both the photon energy and the energy width are selected by use of a multi-channel analyzer, and the number of photons is counted by a counter card. For performing energy discrimination, a low-dose-rate X-ray generator for photon counting was developed; the maximum tube voltage and the minimum tube current were 110 kV and 1.0 microA, respectively. In energy-discrimination CT, the tube voltage and the current were 60 kV and 20.0 microA, respectively, and the X-ray intensity was 0.735 microGy/s at 1.0 m from the source and with a tube voltage of 60 kV. Demonstration of enhanced iodine K-edge X-ray CT was carried out by selection of photons with energies just beyond the iodine K-edge energy of 33.2 keV.

Embossed radiography utilizing energy subtraction.

Currently, it is difficult to carry out refraction-contrast radiography by using a conventional X-ray generator. Thus, we developed an embossed radiography system utilizing dual-energy subtraction for decreasing the absorption contrast in unnecessary regions, and the contrast resolution of a target region was increased by use of image-shifting subtraction and a linear-contrast system in a flat panel detector (FPD). The X-ray generator had a 100-microm-focus tube. Energy subtraction was performed at tube voltages of 45 and 65 kV, a tube current of 0.50 mA, and an X-ray exposure time of 5.0 s. A 1.0-mm-thick aluminum filter was used for absorbing low-photon-energy bremsstrahlung X-rays. Embossed radiography was achieved with cohesion imaging by use of the FPD with pixel sizes of 48 x 48 microm, and the shifting dimension of an object in the horizontal direction ranged from 100 to 200 microm. At a shifting distance of 100 mum, the spatial resolutions in the horizontal and vertical directions measured with a lead test chart were both 83 microm. In embossed radiography of non-living animals, we obtained high-contrast embossed images of fine bones, gadolinium oxide particles in the kidney, and coronary arteries approximately 100 microm in diameter.

X-ray fluorescence camera for imaging of iodine media in vivo.

X-ray fluorescence (XRF) analysis is useful for measuring density distributions of contrast media in vivo. An XRF camera was developed for carrying out mapping for iodine-based contrast media used in medical angiography. Objects are exposed by an X-ray beam from a cerium target. Cerium K-series X-rays are absorbed effectively by iodine media in objects, and iodine fluorescence is produced from the objects. Next, iodine Kalpha fluorescence is selected out by use of a 58-microm-thick stannum filter and is detected by a cadmium telluride (CdTe) detector. The Kalpha rays are discriminated out by a multichannel analyzer, and the number of photons is counted by a counter card. The objects are moved and scanned by an x-y stage in conjunction with a two-stage controller, and X-ray images obtained by iodine mapping are shown on a personal computer monitor. The scan pitch of the x and y axes was 2.5 mm, and the photon counting time per mapping point was 2.0 s. We carried out iodine mapping of non-living animals (phantoms), and iodine Kalpha fluorescence was produced from weakly remaining iodine elements in a rabbit skin cancer.

Intravenous injection of phagocytes transfected ex vivo with FGF4 DNA/biodegradable gelatin complex promotes angiogenesis in a rat myocardial ischemia/reperfusion injury model.

Conventional gene therapies still present difficulties due to poor tissue-targeting, invasiveness of delivery, method, or the use of viral vectors. To establish the feasibility of using non-virally ex vivo transfected phagocytes to promote angiogenesis in ischemic myocardium, gene-transfection into isolated phagocytes was performed by culture with positively charged gelatin impregnated with plasmid DNA. A high rate of gene transfection was achieved in rat macrophages and human monocytes, but not in mouse fibroblasts. The efficiency was 68 +/- 11% in rat macrophages and 78 +/- 8% in human monocytes. Intravenously injected phagocytes accumulated predominantly in ischemic tissue (13 +/- 8%) and spleen (84 +/- 6%), but negligibly in other organs in rodents. The efficiency of accumulation in the target ischemic tissue reached more than 86% on direct local tissue injection. In a rat model of myocardial ischemia-reperfusion, intravenous injection of fibroblast growth factor 4 (FGF4)-gene-transfected macrophages significantly increased regional blood flow in the ischemic myocardium (78 +/- 7.1 % in terms of flow ratio of ischemic/non-ischemic myocardium) compared with intravenous administration of saline (36 +/- 11%) or nontransfected macrophages (42 +/- 12 %), or intramuscular administration of naked DNA encoding FGF4 (75 +/- 18 %). Enhanced angiogenesis in the ischemic tissue we confirmed histologically. Similarly, intravenous injection of FGF4-gene-transfected monocytes enhanced regional blood flow in an ischemic hindlimb model in mice (93 +/- 22 %), being superior to the three other treatments described above (38 +/- 12, 39 +/- 15, and 55 +/- 12%, respectively). Phagocytes transfected ex vivo with FGF4 DNA/gelatin promoted angiogenesis. This approach might have potential for non-viral angiogenic gene therapy.

Less-invasive method for microsphere delivery to coronary circulation and evaluation of myocardial ischemic change in small animals.

We developed a less-invasive method for semi-selective administration of drugs into coronary arteries in small animals. With this method, we created microvascular myocardial ischemia in rabbits by microsphere injection. A 4F catheter was inserted into the left ventricle via the right common carotid artery and a balloon catheter into the descending thoracic aorta. Microspheres were administered into the left ventricle with temporary occlusion of the descending aorta and carotid arteries. In these conditions, regional blood flow in the heart was 10.8-times as much as in the kidney. Seventeen days after microsphere injection, the contractile function of the heart muscle deteriorated and the left ventricular endodiastolic pressure was increased. Patchy NADH-fluorescence was observed all over the left ventricular myocardium. Myocardial lactate concentration was higher than the normal standard animals. Histological analysis revealed that microscopic patchy necrosis was noted only in the myocardium but not in other organs. Semi-selective delivery of recombinant adenovirus expressing lacZ using the same method induced a gene expression in the heart. Thus, a unique model for microvascular myocardial ischemia was created by semi-selective delivery of microspheres into the coronary artery without special technique or equipment. The present model is also applicable to semi-selective gene transfer to the heart.

Search for appropriate experimental methods to create stable hind-limb ischemia in mouse.

OBJECTIVE: Stable animal models for refractory peripheral arterial disease are not established. A standardized animal model of hind-limb ischemia is required upon searching effective treatment for this condition. The aim of the study is to verify previously used hind-limb ischemia models to find a standard method. METHODS: Using Balb/ca mice six various methods of inducing hind-limb ischemia were applied and two weeks after operation degree of ischemic damage were examined. Six methods include V group, A group, AV group, A-strip group, AV-strip group and Prox-A group (refer the text). RESULTS: Degree of ischemia was evaluated macroscopically by judging toes, foot, knee, and total hind-limb necrosis. We found that severity of damage was markedly different among different methods. Furthermore the severity of necrosis was not uniform even in the same method group. CONCLUSIONS: The A-strip group in which the femoral artery from the bifurcation of the deep femoral artery to the saphenous artery was stripped appears to be suitable as a stable severe ischemia model. The A group in which the femoral artery were cut just below the bifurcation of the deep femoral artery appears to be suitable as a chronic mild ischemia model.

Regulation mode of evaporative cooling underlying a strategy of the heat-tolerant FOK rat for enduring ambient heat.

Compared with other rat strains, the inbred FOK rat is extremely heat tolerant. This increased heat tolerance is due largely to the animal's enhanced saliva spreading abilities. The aims of the present study were to 1) quantify the heat tolerance capacity of FOK rats and 2) determine the regulatory mode of the enhanced salivary cooling in these animals. Various strains of rats were acutely exposed to heat. In the heat-intolerant strains, saliva spreading was insufficient and the core temperature (Tc) rose rapidly. In contrast, FOK rats maintained an elevated Tc plateau (39.5 +/- 0.7 degrees C) for 5-6 h over a wide range of ambient temperatures (Ta) (37.5-42.5 degrees C). In hot environments the FOK rats secreted copious amounts of saliva and spread it over more than the entire ventral body surface. FOK rats had a low Tc threshold for salivation, and the salivation rate increased linearly in proportion to the Tc deviation from the threshold. No strain difference or temperature effect was observed in the saliva secretion rate from in vitro submandibular glands perfused by sufficient doses of ACh. These results suggest that 1) the ability of FOK rats to maintain a moderate steady-state hyperthermia (39.5 +/- 0.7 degrees C) over a wide Ta range is enabled by a lowered threshold Tc for salivation and functional negative-feedback control of saliva secretion and 2) strain differences in ability to endure heat stress are mainly attributable to changes in the thermoregulatory control system rather than altered secretory abilities of the salivary glands.