Adhesion patterning by a novel air-lock technique enables localization and in-situ real-time imaging of reprogramming events in one-to-one electrofused hybrids.

Although fusion of somatic cells with embryonic stem (ES) cells has been shown to induce reprogramming, single-cell level details of the transitory phenotypic changes that occur during fusion-based reprogramming are still lacking. Our group previously reported on the technique of one-to-one electrofusion via micro-slits in a microfluidic platform. In this study, we focused on developing a novel air-lock patterning technique for creating localized adhesion zones around the micro-slits for cell localization and real-time imaging of post fusion events with a single-cell resolution. Mouse embryonic fibroblasts (MEF) were fused individually with mouse ES cells using a polydimethylsiloxane (PDMS) fusion chip consisting of two feeder channels with a separating wall containing an array of micro-slits (slit width ∼3 µm) at a regular spacing. ES cells and MEFs were introduced separately into the channels, juxtaposed on the micro-slits by dielectrophoresis and fused one-to-one by a pulse voltage. To localize fused cells for on-chip culture and time-lapse microscopy, we implemented a two-step approach of air-lock bovine serum albumin patterning and Matrigel coating to create localized adhesion areas around the micro-slits. As a result of time-lapse imaging, we could determine that cell division occurs within 24 h after fusion, much earlier than the 2-3 days reported by earlier studies. Remarkably, Oct4-GFP (Green Fluorescent Protein) was confirmed after 25 h of fusion and thereafter stably expressed by daughter cells of fused cells. Thus, integrated into our high-yield electrofusion platform, the technique of air-lock assisted adhesion patterning enables a single-cell level tracking of fused cells to highlight cell-level dynamics during fusion-based reprogramming.

Microorifice-based high-yield cell fusion on microfluidic chip: electrofusion of selected pairs and fusant viability.

Microorifice-based fusion makes use of electric field constriction to assure high-yield one-to-one fusion of selected cell pairs. The aim of this paper is to verify feasibility of high-yield cell fusion on a microfluidic chip. This paper also examines viability of the fusant created on the chip. We fabricated a microfluidic chip to fuse selected cell pairs and to study postfusion behavior. We used a self-forming meniscus-based fabrication process to create microorifice with a diameter of 2-10 microm on the vertical walls in a microfluidic channel. When 1 MHz was applied to electrodes located on both sides of the microorifice, dielectrophoretic force attracted the cells toward microorifice to form a cell pair. Once the cells get into contact, fusion pulse was applied. Real time imaging of cells during fusion and cytoplasmic dye transfer between cells indicated success of cell fusion. We found that when high frequency voltage for dielectrophoresis was swept from 1 MHz to 10 kHz in 100 micros, cell fusion was initiated. The effective electric field strength was 0.1-0.2 kV/cm. We analyzed viability by imaging fusant going into cell division phase after 48 h of incubation. We conclude that fabricated microfluidic chip is suitable for high-yield one-to-one fusion and creation of viable fusants. This technology should be a useful tool to study fusion phenomena and viability of fusants, as it allows imaging of the cells during and after the fusion.

Polarisation and membrane voltage of ellipsoidal particle with a constant membrane thickness: a series expansion approach.

The estimation of the membrane voltage and the polarisation factor of biological cells provide a base for the study of bio-manipulation techniques, such as dielectrophoresis, electroporation or electrofusion. To model a biological cell, an ellipsoidal particle with an insulating membrane is sometimes employed, but due to the limitation of the confocal nature of the coordinate system, the membrane thickness is assumed to vary with the position, despite the fact that the lipid bilayer membrane has a uniform thickness. The authors present a method to rigorously treat the uniform-thickness condition in a system having an axial symmetry. The method is based on the harmonic expansion of the field, to include the condition of the uniform-membrane thickness as a series expansion of the geometrical factor, and to solve the field problem as an interaction of the harmonic components. The conventional variable thickness model has been identified as being equivalent to neglecting the harmonic interactions in the uniform-thickness model. Numerical calculations are done of the membrane voltage and the polarisation factor, and it has been found that the discrepancy between the proposed rigorous model and the conventional variable thickness model becomes significant when field deformation is large due to the high axial ratio of the ellipsoid.

High-yield electrofusion of biological cells based on field tailoring by microfabricated structures.

The authors present the use of electric-field constriction created by a microfabricated structure to realise high-yield electrofusion of biological cells. The method uses an orifice on an electrically insulating wall (orifice plate) whose diameter is as small as that of the cells. Owing to the field constriction created by the orifice, we can induce the controlled magnitude of membrane voltage selectively around the contact point, regardless of the cell size. The field constriction also ensures 1:1 fusion even when more than two cells are forming a chain at the orifice. A device for electrofusion has been made with a standard SU-8 lithography and PDMS molding, and real-time observation of the electrofusion process is made. Experiments using plant protoplasts or mammalian cells show that the process is highly reproducible, and the yield higher than 90% is achieved.

The activity ratio of the cytosolic MDH/LDH and the isoenzyme pattern of LDH in the peripheral leukocytes of dogs, cats and rabbits.

The activities of malate dehydrogenase (MDH) and lactate dehydrogenase (LDH) and the pattern of the isoenzymes of LDH were determined in the peripheral blood leukocytes of dogs, rabbits and cats. Rabbits had significantly higher plasma glucose concentrations than dogs or cats. Feline leukocytes showed higher LDH and lower MDH activities than canine or rabbit leukocytes. The M/L ratio, defined as the MDH activity divided by the LDH activity in cytosolic fractions, was considered to be a good indicator with which to evaluate the metabolic state in animal tissues. The M/L ratio was highest in canine and lowest in feline leukocytes. LDH-2 and LDH-3 isoenzymes were dominant in canine leukocytes. LDH-1 and LDH-2 were dominant in rabbit leukocytes, whereas LDH-5 was dominant in feline leukocytes. It was evident that there were significant differences in energy metabolism between the leukocytes of dogs, rabbits and cats.

Metastatic calcinosis circumscripta treated with an oral charcoal absorbent in a dog.

A five-year-old West Highland white terrier dog was admitted to the teaching hospital of Nippon Veterinary and Animal Science University due to swelling and pain of the foot pads. Examinations revealed that the dog had renal failure and calcinosis circumscripta on its foot pads. The diagnosis was metastatic calcinosis circumscripta secondary to renal failure. An oral charcoal adsorbent (Kremezin) was used to treat this condition. Following this treatment, a significant decrease in the Ca x P value (the serum calcium level x the serum phosphorus level) was observed, and the dog's condition improved dramatically. This case suggests that charcoal adsorbent (Kremezin) may be useful for treating metastatic calcinosis circumscripta in dogs.

Visualization of a specific sequence on a single large DNA molecule using fluorescence microscopy based on a new DNA-stretching method.

A method for analyzing large DNA which makes it possible to obtain spatial information on the positions of specific sequences along a DNA molecule has been developed. Making use of the fact that large DNA molecules are stably elongated under an alternating-current field in a concentrated linear polymer solution, the direct observation of elongated individual lambda DNA molecules with fluorescence probes was carried out using fluorescence microscopy. The spatial positions of the fluorescent spots of the probe (fluorescence-labeled restriction endonuclease EcoRI) on DNA molecules were determined by image analysis. As expected, fluorescent spots of EcoRI were observed at certain positions on lambda DNA, where sequences to which EcoRI binds are located. Finally, the potential application of single large DNA molecule analysis using this DNA-stretching method is discussed.

Comparison of the activities of enzymes related to glycolysis and gluconeogenesis in the liver of dogs and cats.

Activities of enzymes related to glucose metabolism were measured in canine and feline liver. There were no significant differences in plasma glucose and immunoreactive insulin concentrations between dogs and cats. Glucokinase activities were absent in feline liver, however, activities of other glycolytic enzymes such as hexokinase, phosphofructokinase and pyruvate kinase, were significantly higher than those in canine livers. Activities of rate limiting enzymes of gluconeogenesis such as pyruvate carboxylase, fructose-1, 6-bisphosphatase and glucose-6-phosphatase in feline livers were significantly higher than those in canine livers.

Direct measurement of DNA by means of AFM.

We have demonstrated that the electrostatic stretch-and-positioning method is useful for the analysis of a long DNA molecule by means of atomic force microscopy (AFM). DNA molecules were stretched parallel to the field line, and immobilized onto the aluminum electrodes patterned on a glass plate. Through AFM observation, we confirmed the immobilization of individual DNA molecules, not aggregate.

Decreased pulmonary arterial endothelium-dependent relaxation in heartworm-infected dogs with pulmonary hypertension.

OBJECTIVE: To investigate the contractility of pulmonary arterial smooth muscle and the relation between pulmonary hypertension and endothelium-derived relaxing factor in canine heartworm disease. ANIMALS: 18 noninfected control and 9 heartworm-infected dogs. PROCEDURE: Mean pulmonary arterial blood pressure was measured in vivo, and tension of pulmonary arterial strips was measured by use of the isometric tension method. RESULTS: After phenylephrine (10(-5)M)-induced contraction of the pulmonary vascular smooth muscle, carbamylcholine chloride (CCh, 10(-6)M) caused more relaxation of the vascular smooth muscle of noninfected, dogs than that of heartworm-infected dogs. Furthermore, the degree of CCh-induced relaxation was inversely correlated with mean pulmonary arterial blood pressure in the noninfected and the heartworm-infected dogs. The CCh-induced relaxation was inhibited by pretreatment with NG-nitro-L-arginine methyl ester hydrochloride (10(-5)M), and in reversed dose-dependent manner by L-arginine (10(-4) to 3 x 10(-2)M). Sodium nitroprusside (10(-8) to 10(-5)M) caused a dose-dependent relaxation in all vessels, and there was no significant difference in the relaxation responses in both groups except at 10(-7)M for vessels with intact endothelium from noninfected dogs. CONCLUSION: The depression of endothelium-dependent relaxation is correlated with the pulmonary arterial blood pressure in heartworm-infected dogs, suggesting that the decrease is one of the essential factors for the genesis of pulmonary hypertension in canine filariasis.

Changes in glucose transport activities in mammary adenocarcinoma of dogs.

The activities of D-glucose transport (D-GT) and cytosolic enzymes were significantly higher in mammary adenocarcinoma of dogs than in mammary gland from normal dogs. The activities of D-GT in adenocarcinoma were over three-and-a-half times higher than in the controls. The K(m) value of the D-GT activity for glucose in both the adenocarcinoma and normal mammary gland was approximately 0.9 mM. The activities of the key glycolytic enzymes, hexokinase and pyruvate kinase, in the adenocarcinoma were also more than three-and-a-half times higher than in the controls. The increased activities of D-GT are considered to be accompanied by an acceleration of glucose utilisation in the adenocarcinoma of dogs.

Multifocal ventricular tachycardia after resuscitation by direct current counter shock in a dog.

Cardiac arrest occurred in a male Labrador Retriever dog weighing 27.8 kg during induction to anesthesia. Immediately after the failure of resuscitation by the external cardiac compression, thoracotomy was performed and open chest direct current (DC) counter shocks were applied with routine emergency medications. Then the dog recovered consciousness. Although cardiac rhythm just after resuscitation was sinus tachycardia with paroxysmal supraventricular tachycardia, multifocal ventricular arrhythmia occurred 2 hr after resuscitation. This arrhythmia might be the result from reversible cardiac lesions due to DC counter shock.

Longitudinal distribution of pulmonary vascular compliance in dogs.

The longitudinal distribution of pulmonary vascular compliance was evaluated in isolated canine lung lobes using arterial-(AO), venous-(VO), and double-occlusion (DO) techniques. Total vascular compliance (Ctau) was separated into pulmonary arterial (Ca) and venous compliance (Cv) in lumped model of pulmonary circulation. Under constant pulmonary venous pressure (Pv) at 5 mmHg, blood inflow to the lobe (Q) was gradually increased by changing pulmonary arterial pressure (Pa) from 10 to 22 mmHg at 4 mmHg ranges. Changes in vascular blood volume (deltaV) with each increment in Q were determined by decreased reservoir blood volume of perfusion system. DO was performed at each level of Q and allowing all vascular pressures to equilibrate at the same static pressure (Ps), which was equal to the compliance-weighted average pressure in the circulation. Ctau was obtained from the slope of the relationship between Ps and deltaV. When Pa and Pv were 14 and 5 mmHg, AO, VO, and DO were performed to measure pressures at Ca (Pca) and Cv (Pcv) and Ps. The arterial-to-venous compliance ratio (Ca/Cv) was evaluated using Pca, Pcv, and Ps measurements. Ctau was 0.113 +/- 0.012 ml/kg/mmHg. Ca/Cv was 0.30. Ca and Cv were 0.026 +/- 0.013 and 0.087 +/- 0.007 ml/kg/mmHg, respectively. These data demonstrated the usefulness of AO, VO, and DO techniques in evaluating the longitudinal distribution of compliance in canine pulmonary vasculature.

The effect of hypovolemic shock and reperfusion on the hepatic oxygen supply-uptake relationship in the dog.

The hepatic oxygen supply-uptake relationship was investigated during hypovolemic shock using a right heart bypass technique. The results were dissimilar to those previously reported in that the ratio of liver oxygen delivery to systemic oxygen delivery was significantly decreased during shock. The decreased ratio was due to a significant decrease in the portal venous oxygen delivery when compared to the decrease in the systemic oxygen delivery. The decrease in portal venous oxygen delivery was caused not only by the decrease in portal venous blood flow, but also by the decrease in oxygen content of portal blood. The ratio of hepatic arterial oxygen delivery, on the other hand, was significantly increased during shock. Hypovolemic shock increased the liver oxygen extraction ratio to nearly 100% of the pre-shock value. These findings suggest a hepatic protective mechanism for matching oxygen uptake to rising hepatic oxygen requirements. Liver oxygen delivery returned to pre-shock value after correction of hypovolemia primarily due to a significant increase in hepatic arterial oxygen delivery. A significant negative correlation between the liver oxygen extraction ratio and the oxygen content of hepatic venous blood was observed. The hepatic venous oxygen content appears to be a simple and appropriate index of liver oxygenation in clinical medicine because it is difficult to evaluate the liver oxygen extraction ratio directly.

Effects of hypovolemic shock and reperfusion on liver blood flow in the dog.

Liver blood flow was investigated in hypovolemic shock using a modified right heart bypass technique which can obtain accurate portal blood flow. Findings were similar to those previously reported: hepatic blood flow accounted for 34% of cardiac output in this study; 76% of hepatic blood flow was delivered from the portal vein and 24% from the hepatic artery. Hypovolemic shock markedly decreased total liver blood flow by a reduction in portal venous blood flow. The findings of this study provide evidence that mesenteric blood flow is a peripheral circulation circuit where blood flow is restricted during reduced circulatory volume. Development of a hepatic arterial buffer response during hypovolemic shock was confirmed by an increased ratio of hepatic arterial flow to cardiac output. Reduced total hepatic blood flow during hypovolemic shock returned to control flow by an increase in hepatic arterial flow after reperfusion. The results of this study demonstrate that compensated reactions for maintaining liver blood flow mainly due to the hepatic arterial buffer response were functioned both during hypovolemic shock and after elimination of shock.

The effect of indomethacin on liver blood flow and oxygen supply-uptake relationship in the dog.

The effects of indomethacin on liver blood flow and oxygen supply-uptake relationship were investigated using a right heart bypass technique. Portal venous blood flow was decreased by the mesenteric vascular effects of indomethacin, which produce intense mesenteric vasoconstriction. Hepatic arterial blood flow was increased and therefore, total liver blood flow was not significantly changed after indomethacin administration. Portal venous oxygen delivery was significantly decreased by reductions in both portal venous blood flow and portal venous oxygen content. Total liver oxygen delivery, however, was not changed after indomethacin administration. This response was caused by a large increase in hepatic arterial oxygen delivery. Liver oxygen uptake and liver oxygen extraction ratio were not changed after indomethacin administration. We conclude, therefore, that total liver blood flow and oxygen delivery were well maintained, even if the mesenteric vascular effects of indomethacin decreased both portal venous blood flow and portal venous oxygen delivery.

The effects of dopamine and dobutamine on liver oxygen supply-uptake relationship in the dog.

The effects of dopamine and dobutamine in various doses on liver oxygen supply-uptake relationship were studied in 12 mongrel dogs. Dopamine 3 and 7 micrograms/kg/min infusion rates and dobutamine 5 micrograms/kg/min infusion rate did not produce any changes in total liver oxygen delivery. On the contrary, total liver oxygen delivery was increased at the 15 micrograms/kg/min dopamine in fusion rate and dobutamine 10 and 15 micrograms/kg/min infusion rates. The ratio of total liver oxygen delivery to the systemic oxygen delivery was increased at the 15 micrograms/kg/min dopamine infusion rate. Liver oxygen extraction ratio was decreased at the 15 micrograms/kg/min dopamine infusion rate and at the same rate of dobutamine. These decreases were due to the increases in oxygen delivery while both oxygen uptakes were avariant from control levels. The results of this study demonstrated that high dose of dopamine (15 micrograms/kg/min) and medium and high doses of dobutamine (10 and 15 micrograms/kg/min) should be useful to increase the liver oxygen delivery. However, these increases in liver oxygen delivery during dopamine and dobutamine infusion were not associated with an improvement in liver oxygen metabolism, since liver oxygen uptake was not changed.

The selective effects of dopamine and dobutamine on liver circulation in the dog.

The selective effects od dopamine and dobutamine in various doses on liver circulation were studied in 12 mongrel dogs. Dopamine increased portal flow but decreased hepatic arterial flow markedly as infusion rate of dopamine increased. Dopamine 3 micrograms/kg/min infusion rate produced vasodilation in mesenteric vascular bed and the portal flow ratio to cardiac output was significantly increased. Dobutamine increased both portal and hepatic arterial flows at the 5 and 10 micrograms/kg/min dobutamine infusion rates, and decreased hepatic arterial flow at the 15 micrograms/kg/min dobutamine infusion rate. Both dopamine and dobutamine increased total liver flows, however, total liver flow ratio to cardiac output was not increased. Pressure gradient of portal system was not changed during dopamine and dobutamine infusion, since both portal venous pressure and hepatic venous pressure were avariant from control values. These findings suggest that congestive hyperemia was not occurred in intrahepatic portal vascular system when portal flows were increased during dopamine and dobutamine infusion. The results of this study demonstrate that both dopamine and dobutamine did not produce selective increases in total liver blood flow. In addition, both agents should be safe to use to the normal liver patient; total liver blood flow did not decrease and intrahepatic congestive hyperemia was not occurred when portal flow was increased.

Effect of food on liver circulation in conscious dog.

Hepatic hemodynamics and the liver oxygen supply-uptake relationship, in response to eating, were investigated in a chronically catheterized conscious dog method. Portal venous pressure was significantly increased after eating, however was within the normal range reported previously. Hepatic venous pressure correlated well with portal venous pressure throughout the experiment, therefore, the pressure gradient of the portal system was unchanged. Hepatic venous oxygen content, correlated well with liver oxygen extraction, was unchanged after eating. Therefore, it is possible to assume that liver oxygen supply-uptake relationship is well maintained during digestion of food.

Regression of hypertrophic osteopathy following removal of intrathoracic neoplasia derived from vagus nerve in a dog.

Surgical removal of an intrathoracic tumor derived from a vagus nerve was undergone in a dog with hypertrophic osteopathy. The tumor was pathologically diagnosed as malignant schwanoma. Soft tissue swelling, lameness, and itchiness in four limbs disappeared within 7 days after surgery. The proliferated periosteal osteophytes of the four limbs was progressively reduced with time by follow-up radiography on the 58th day after surgery. On the 710th day after surgery, these osteophytes were greatly decreased as osteopathy, malignant schwanoma.