BACKGROUND: Flow cytometric analysis is the standard method for enumerating CD34+ stem cells in hematopoietic stem cell transplantation. However, it has some limitations such as expensive instrumentation, high reagent costs, and discrepancies between technicians and laboratories. We compared counts of total nucleated cells (TNCs) and CD34+ cells counts obtained from a flow cytometer with a newly-developed image-based microscopic cell counter (ADAM II) to evaluate the possibility of clinical application of the ADAM II.METHODS: We used 18 samples of circulating peripheral blood (PB) and waste tube fractions of peripheral blood stem cells (PBSCs) harvested by apheresis after G-CSF mobilization from adult volunteer donors. We assessed the reproducibility and linearity of the new procedure and compared the numbers of TNCs and viable CD34+ cells determined with the ADAM II and two different flow cytometers (FACSCalibur, FACSCanto II).RESULTS: Numbers of viable CD34+ cells determined with the ADAM II were accurate over the expected range; the intra-assay coefficient of variation was ≤19.8%. Linearity was also satisfactory (R²=0.99). TNC counts obtained with the ADAM II were highly correlated with those obtained with the FACSCalibur (R²>0.9841, P<0.0001) and FACSCanto II (R²>0.9620, P<0.0001), as were the numbers of viable CD34+ cells obtained with the ADAM II and the FACSCalibur and FACSCanto II (R²>0.9911, P<0.0001 and R²>0.9791, P<0.0001), respectively.CONCLUSION: The newly developed image-based microscopic cell counter (ADAM II) appears to be suitable for enumerating TNCs and viable CD34+ cells.
Adult ; Blood Component Removal ; Cell Count ; Granulocyte Colony-Stimulating Factor ; Hematopoietic Stem Cell Transplantation ; Humans ; Methods ; Stem Cells ; Tissue Donors ; Volunteers

BACKGROUND AND OBJECTIVES: Several treatment options have been introduced to control Meniere's disease (MD). Among them, intratympanic injection of gentamicin (ITGM) is now accepted as a standard treatment method for intractable vertigo in patients with hearing impaired MD. The purpose of this study was to analyze the clinical course and treatment flow in patients with MD and to analyze the efficacy and problems after ITGM. SUBJECTS AND METHOD: A retrospective study was performed on 458 patients who were diagnosed as definite MD from May 2003 to October 2007 and were followed up at least 1 year. The authors investigated the course of disease and the results in our patients according to the treatment options. Several clinical parameters including frequency of vertigo, audiometry, caloric and rotation test were reviewed and analyzed according to the guideline of the AAO-HNS (1995), if necessary. Efficacy and problems after ITGM were reviewed. RESULTS: Among 458 patients, vertigo was resolved or improved in 399 patients (87.1%) with medical management. Fifty-nine patients needed further treatment (endolymphatic sac decompression: 4, Meniette: 2, ITGM: 53). Among 53 patients who got ITGM, 40 patients were followed up for more than 1 year after injection. Thirty-six patients (87.5%) showed successful control of vertigo. The average pure-tone threshold was changed from 54.1 dB to 56.9 dB after treatment. Three patients (7.5%) revealed more than 20 dB aggravation. CONCLUSION: Vertigo was controlled by supportive treatments or medication in 87% of definite MD patients. And ITGM could effectively and reasonably control vertigo for intractable MD patients.
Audiometry ; Endolymphatic Hydrops ; Gentamicins ; Hearing ; Humans ; Meniere Disease ; Retrospective Studies ; Vertigo

BACKGROUND: Recent studies suggest that the cardioprotective effect of ischemic preconditioning (IPC) is related to intracellular glycogen content in rat hearts, however, controversies still remain. METHODS: To test this hypothesis, isolated Langendorff-perfused rabbit hearts were subjected to 45 min global ischemia followed by 120 min reperfusion with IPC (n=0) or without IPC (ischemic control, n=). IPC was induced by one cycle of 5 min global ischemia and 10 min reperfusion. In the glucose (G)-free preconditioned group (n=0), G depletion-repletion was induced by perfusion with G-free Tyrode solution for 5 min and then G-containing Tyrode solution for 10 min followed by 45 min ischemia and 120 min reperfusion. For glycogen depletion or loading, hearts were treated with sodium acetate (NA, 5 mM, n=) or insulin (Ins, 1 unit/L, n=) for 15 min before 45 min ischemia. Left ventricular function and coronary flow (CF) were continuously recorded during experiments. Myocardial cytosolic and membrane protein kinase C (PKC) activities were measured by 32P-gamma-ATP incorporation into PKC-specific pepetide; glycogen content in the cardiac myocytes was determined by spectrophotometry with amyloglucosidase; expression of PKC isozymes was determined by Western blot with monoclonal antibodies. Infarct size was determined by staining with tetrazolium salt and planimetry. Data were analyzed by ANOVA and Tukey's post-hoc test. RESULTS: IPC or G-free preconditioning enhanced LV functional recovery; NA did not influence on functional recovery but Ins depressed it. Infarct size was significantly reduced by IPC, G-free preconditioning, and NA treatment (35.3+/-2.1% in the ischemic control, 18.7+/-1.2% in the IPC, 22.1+/-1.2% in the G-free preconditioned, 16.3+/-1.2% in the NA-treated group, and 32.8+/-1.6% in the Ins-treated group, p<0.05). Membrane PKC activities significantly increased by IPC, IPC and 45 min ischemia, G-free preconditioning, and G-free preconditioning and 45 min ischemia; especially, expression of membrane PKC-epsilon increased by IPC and G-free preconditioning. Glycogen content decreased by 45 min ischemia, IPC, G-free preconditioning, and by NA treatment, but increased by Ins treatment. CONCLUSION: These results suggest that in rabbit heart, intracellular glycogen may not significantly be related with the cardioprotective effect of IPC; G-free preconditioning could not improve post-ischemic contractile dysfunction but it has an infarct size-limiting effect; this cardioprotective effect may be related in part to activation of PKC, especially epsilon isozyme.
Animals ; Antibodies, Monoclonal ; Blotting, Western ; Cytosol ; Glucan 1,4-alpha-Glucosidase ; Glucose ; Glycogen* ; Heart ; Insulin ; Ischemia ; Ischemic Preconditioning* ; Isoenzymes ; Membrane Proteins ; Membranes ; Myocytes, Cardiac ; Perfusion ; Phosphotransferases ; Protein Kinase C* ; Protein Kinases* ; Rats ; Reperfusion ; Sodium Acetate ; Spectrophotometry ; Ventricular Function, Left

BACKGROUND: Recent studies have suggested that the cardioprotective effect of ischemic preconditioning (IP) is closely related to glycogen depletion and attenuation of intracellular acidosis. In the present study, the authors tested this hypothesis by perfusion isolated rabbit hearts with glucose(G) is closely related to glycogen depletion and attenuation of intracellular acidosis. In the present study, the authors tested this hypothesis by perfusion isolated rabbit hearts with glucose(G)-free perfusate. MATERIAL AND METHOD: Hearts isolated from New Zealand white rabbits (1.5~2.0 kg body weight) were perfused with Tyrode solution by Langendorff technique. After stabilization of baseline hemodynamics, the hearts were subjected to 45 min global ischemia followed by 120 min reperfusion with IP(IP group, n=13) or without IP(ischemic control group, n=10). IP was induced by single episode of 5 min global ischemia and 10 min reperfusion. In the G-free preconditioned group(n=12), G depletion was induced by perfusionwith G-free Tyrode solution for 5 min and then perfused with G-containing Tyrode solution for 10 min; and 45 min ischemia and 120 min reperfusion. Left ventricular functionincluding developed pressure(LVDP), dP/dt, heart rate, left ventricular end-distolic pressure (LVEDP) and coronary flow (CF) were measured. Myocardial cytosolic and membrane PKC activities were measured by 32P-gamma-ATP incorporation into PKC-specific peptide and PKC isozymes were analyzed by Western blot with monoclonal antibodies. Infarct size was determined by staining with TTC (tetrazolium salt) and planimetry. Data were analyzed by one-way analysis of variance (ANOVA) and Turkey's post-hoc test. RESULT: In comparison with the ischemic control group, IP significantly enhanced functional recovery of the left ventricle; in contrast, functional significantly enhanced functional recovery of the left ventricle; in contrast, functional recovery were not significantly different between the G-free preconditioned and the ischemic control groups. However, the infarct size was significantly reduced by IP or G-free preconditioning (39+/-2.7% in the ischemic control, 19+/-1.2% in the IP, and 15+/-3.9% in the G-free preconditioned, p<0.05). Membrane PKC activities were increased significantly after IP (119%), IP and 45 min ischemia(145%), G-free [recpmdotopmomg (150%), and G-free preconditioning and 45 min ischemia(127%); expression of membrane PKC isozymes, alpha and beta, tended to be increased after IP or G-free preconditioning. CONCLUSION: These results suggest that in isolated Langendorff-perfused rabbit heart model, G-free preconditioning (induced by single episode of 5 min G depletion and 10 min repletion) colud not improve post-ischemic contractile dysfunction(after 45-minute global ischemia); however, it has an infarct size-limiting effect.
Acidosis ; Antibodies, Monoclonal ; Blotting, Western ; Cytosol ; Glucose ; Glycogen* ; Heart ; Heart Rate ; Heart Ventricles ; Hemodynamics ; Ischemia ; Ischemic Preconditioning* ; Isoenzymes ; Membranes ; Perfusion ; Rabbits ; Reperfusion

BACKGROUND: We tested recent evidences that IP triggers selective activation of protein kinase C (PKC) isozymes using isolated Langendorff-perfused rabbit heart with PKC activator, phorbol ester (PMA, 0.01 nM) or inhibitor (calphostin C, 200 nM). METHODS: After stabilization of baseline hemodynamics, the hearts were subjected to 45 min global ischemia (I) followed by 120 min reperfusion (R) with IP (IP group, n=18) or without IP (ischemic control group, n=16). IP was induced by single episode of 5 min I and 10 min R. In the PMA-treated group (n=19) and calphostin C-treated preconditioned group (n=15), PMA and calphostin C was given for 5 and 15 min before 45 min I, respectively. Myocardial cytosolic and membrane PKC activities were measured by 32P- -ATP incorporation into PKC-specific pepetide: PKC isozymes were analyzed by Western blot with monoclonal antibodies. RESULTS: IP significantly increased the recovery of the LV function including LVDP and coronary flow (p <0.05):however, enhancement of the functional recovery disappeared by calphostin C or PMA treatment. Cytosolic PKC activity decreased to 82-76% in the IP and PMA-treated group (p <0.05): membrane PKC activity increased to 218-272% (p <0.01). However, both fraction of PKC activity was not changed in the calphostin C-treated preconditioned group. In addition, Western blot revealed that PKC- alpha and epsilon, especially epsilon, were selectively translocated during subsequent sustained ischemia after IP or PMA administration. IP and PMA also reduced infarct size (frim 38 to 10-20%, p <0.05). However, calphostin C blocked infarct reduction effect of IP. CONCLUSION: These results indicate that in isolated rabbit heart model, cardioprotective effect of IP may be related, at least in part, to trigger selective translocation of PKC, especially epsilon isotype.
Antibodies, Monoclonal ; Blotting, Western ; Cytosol ; Heart ; Hemodynamics ; Ischemia ; Ischemic Preconditioning* ; Isoenzymes ; Membranes ; Protein Kinase C* ; Protein Kinases* ; Reperfusion

BACKGROUND: It has been well documented that transient occlusion of the coronary artery causes myocardial ischemia and finally cell death when ischemia is sustained for more than 20 minutes. Extensive studies have revealed that ischemic myocardium cannot recover without reperfusion by adequate restoration of blood flow, however, reperfusion can cause long-lasting cardiac dysfunction and aggravation of structural damage. The author therefore attempted to examine the effect of postischemic reperfusion on myocardial ultrastructure and to determine the rationales for recanalization therapy to salvage ischemic myocardium. MATERIALS AND METHODS: Young Holstein-Friesian cows (130~140 Kg body weight; n=40) of both sexes, maintained with nutritionally balanced diet and under constant conditions, were used. The left anterior descending coronary artery (LAD) was occluded by ligation with 4-0 silk snare for 20 minutes and recanalized by release of the ligation under continuous intravenous drip anesthesia with sodium pentobarbital (0.15 mg/Kg/min). Drill biopsies of the risk area (antero-lateral wall) were performed at just on reperfusion (5 minutes), 1-, 2-, 3-, 6-, 12-hours after recanalization, and at 1-hour assist (only with mechanical respiration and fluid replacement) after 12-hour recanalization. The materials were subdivided into subepicardial and subendocardial tissues. Tissue samples were examined with a transmission electron microscope (Philips EM 300) at the accelerating voltage of 60 KeV. RESULTS: After a 20-minute ligation of the LAD, myocytes showed slight to moderate degree of ultrastructural changes including subsarcolemmal bleb formation, loss of nuclear matrix, clumping of chromatin and margination, mitochondrial destruction, and contracture of sarcomeres. However, microvascular structures were relatively well preserved. After 1-hour reperfusion, nuclear and mitochondrial matrices reappeared and intravascular plugging by polymorphonuclear leukocytes or platelets was observed. However, nucleoli and intramitochondrial granules reappeared within 3 hours of reperfusion and a large number of myocytes were recovered progressively within 6 hours of reperfusion. Recovery was apparent in the subepicardial myocytes and there were no distinct changes in the ultrastructure except narrowed lumen of the microvessels in the later period of reperfusion. CONCLUSIONS: It is likely that the ischemic myocardium could not be salvaged without adequate restoration of coronary flow and that the microvasculature is more resistant to reversible period of ischemia than subendocardium and subepicardium. Therefore, thrombolysis and/or angioplasty may be a rational method of therapy for coronarogenic myocardial ischemia. However, it may take a relatively longer period of time to recover from ischemic insult and reperfusion injury should be considered.
Anesthesia ; Angioplasty ; Biopsy ; Blister ; Body Weight ; Cell Death ; Chromatin ; Contracture ; Coronary Vessels* ; Diet ; Heart* ; Infusions, Intravenous ; Ischemia ; Ligation* ; Microvessels ; Muscle Cells ; Myocardial Ischemia ; Myocardium ; Neutrophils ; Nuclear Matrix ; Pentobarbital ; Reperfusion Injury ; Reperfusion* ; Respiration ; Sarcomeres ; Silk ; SNARE Proteins ; Sodium

BACKGROUND: Brief episodes of coronary blood flow interruption, ischemic preconditioning (IP), following a prolonged ischemia induces myocardial tolerance to ischemia and improves myocardial function during reperfusion by undefined mechanism. Recently, it has been suggested that the signal transduction pathway of the cardiomyocyte itself may involve in this protection. The aims of the present study were : (1) to examine the effect of adenosine in early phase of IP, (2) to define the relationship between the adenosine and protein kinase C(PKC) METHOD AND RESULTS: Heart isolated from New Zealand White rabbit (1.2 - 1.5kg body weight, n=78) were perfused with Tyrode solution by non-recirculating Langendorff technique. After stabilization of baseline hemodynamics, the hearts were subjected to receiving 45min global ischemia (I) and 120min reperfusion (R) with or without IP. IP was induced by a single dose of 5min I and 10min R. A part of the IP hearts, calphostin C (200nmol/L), a PKC inhibitor, was administered 5min before IP and sustained during IP regimen. Left ventricular function and coronary flow were monitored. Infarct size was determined by staining with 1% triphenyltetrazolium chloride solution and computerized planimetry. Adenosine concentration in the coronary flow was determined by HPLC. Myocardial cytosolic and membrane PKC activities were measured by (32)P-r-ATP incorporation into PKC specific peptide. Expression of PKC-e and PKC-o was determined by SDS-PAGE and Western blot. IP enhanced improvement of functional recovery (p<0.05, in the left ventricular developed and end-diastolic pressure ; p<0.01, in the coronary flow) during 120min R after 45min I. Preconditioned hearts showed reduction in the infarct size compared with the non-preconditioned hearts (p<0.05) ; however, IP-induced protection was lost by calphostin C. Adenosine release from the cardiomyocytes abruptly increased to 10-20 folds baseline just after IP manipulation and decreased rapidly on reperfusion. Cytosolic PKC activity significantly decreased in the preconidtioned hearts which received 45min I(p<0.05) and 45min I and 120min R(p<0.01), while the membrane fraction increased in the former(p<0.05) and the latter(p<0.01) groups. There was no significant difference in the PKC-o activity among all experimental groups in cytosolic and membrane fraction, however, the membrane PKC-e isoenzyme activity was increased in the preconditioned hearts which received 45min I. CONCLUSION: These results indicate that (1) a single dose of brief ischemia has an infarctlimiting effect and can improve post-ischemic contractile dysfunction after 45min subsequent sustained I ; and (2) increase of adenosine release in the earlier period of IP regimen and translocation of PKC from the cytosol to myocyte membrane may be important processes signal transduction for protection. These results suggest that cardioprotective mechanism responsible for IP in isolated rabbit heart may be initiated by adenosine and PKC.
Adenosine* ; Blotting, Western ; Body Weight ; Chromatography, High Pressure Liquid ; Cytosol ; Electrophoresis, Polyacrylamide Gel ; Heart ; Hemodynamics ; Ischemia ; Ischemic Preconditioning* ; Membranes ; Muscle Cells ; Myocytes, Cardiac ; New Zealand ; Protein Kinase C* ; Protein Kinases* ; Reperfusion ; Signal Transduction ; Ventricular Function, Left

BACKGROUND: It has been demonstrated that ischemic preconditioning(IP, repetitive brief period of ischemia and reperfusion) enhances recovery of post-ischemic contractile dysfunction and reduces incidences of reperfusion-arrhythmia and infarct size after a prolonged ischemia. A lot of mechanisms have been proposed, however, controversies still remain. Recent studies suggested that IP could activate protein kinase C(PKC). Therefore, we measured left ventricular function, myocardial creatinin and PKC activities, and infarct size to assess whether IPs cardioprotective effect is related to PKC activation using isolated rabbit hearts. METHODS AND RESULTS: Hearts isolated from New Zealand White rabbits(1.5-2.0kg body weight) were perfused with Tyrode solution by Langendorff technique. After stabilization of baseline hemodynamics, the hearts were subjected to 60-min ischemia followed by 120-min reperfusion with IP(IP group, n=15) or without IP(control group, n=14), IP was induced by 4 cycles of 5-min global ischemia and 5-min reperfusion. Left ventricular function including developed pressure(LVEDP), dp/dt, heart rate(HR), and coronary flow(CF) was measured to determine the recovery of LVEDP, RPP(rate-pressure product, HRXLVEDP) and CF to baseline measurement. Frequency of arrhythmia was counted on reperfusion. Myocardial CK-MB, myocardial cytosolic and membrance PKC were measured and the infarct size was determined by staining with tetrazolium salt and planimetry. Data were analyzed by one-way ANOVA, Tukey's post-hoc test and t-test. There was no significant differences in the recovery of LVEDP, dp/dt, RPP, and CF and frequency of arrhythmia during reperfusion between the control and the IP groups. In comparison with the control groups, however, CK-MB was significantly lowered in the IP group(P < 0.05). Cytosolic PKC was significantly decreased but membrance PKC was increased(p < 0.05). These findings indicate that PKC was translocated and activated by IP. Furthermore infarct size was smaller and limited to the antero-lateral or posterior wall and papillary muscle in the IP group(p < 0.05). CONCLUSION: These results indicate that IP dose not improve post-ischemic contractile dysfunction after a prolonged ischemia of 60 minutes but has an infarct-limiting effect. This cardioprotective effect of IP may be related to PKC activation.
Arrhythmias, Cardiac ; Cytosol ; Heart* ; Hemodynamics ; Incidence ; Ischemia ; Ischemic Preconditioning* ; New Zealand ; Papillary Muscles ; Protein Kinase C ; Protein Kinases* ; Reperfusion ; Ventricular Function, Left*

BACKGROUND: Single or multiple episodes of brief period of ischemia and reperfusion(ischemic preconditioning, IP) have been shown to limit infarct size after a subsequent longer period of ischemia. A considerable number of possible mechanisms has been proposed, however, controversies still remain. Accordingly, we evaluated the effect of four cycles of 5 minutes ischemia and 5 minutes reperfusion(IP) followed by subsequent 30 minutes ischemia(ISCH) and 60 minutes reperfusion using isolated Langendorff-Perfused rabbit hearts. Methods and RESULTS: After a 50-minute recovery phase, parameters of the left ventricular function(LVF) including left ventricular developed pressure(LVDP), contractility and the heart rate were recorded, and ultrastructure was examined. Myosin ATPase activity was determined by measurement of inorganic phosphorus and isozymes of the myosin heavy chain were examined by polyacrylamide gel electrophoresis containing pyrophosphate buffer. The ISCH hearts showed severe to irreversible change of the cardiac myocytes homogenously in contrast to the IP hearts in which changes were not homogenous and irreversible injury was only focal. However, parameters of the LVF were not significantly different between the IP and the ISHC hearts during reperfusion. Myosin ATPase activities were also not significantly different(0.67+/-0.123 micromol/mg protein/h in the IP hearts, 0.56+/-0.172 micromol/mg protein/h in the ISCH hearts, and 0.76+/-0.239 micromol/mg protein/h in the control hearts). Band patterns of the myofibrillar proteins, separated by sodium ddodecyl sulfate-polyacrylamide gel electrophoresis, revealed no differences between the IP, ISCH and the control hearts. Myosin heavy chains in the IP and the ISCH hearts were separated into 3 isozymes, V1,V2and V3in pyrophosphate gel electrophoresis in contrast that the control hearts revealed two isozymes, V1and V2. However, there were no differences in the protein composition and electrophoretic motility between the IP and the ISCH hearts. CONCLUSION: These results indicate that IP could not attenuate the changes in LVF, myosin ATPase activity and myosin isozymes on reperfusion, however, it could attenuate the ultrastructural changes of the cardiac myocytes.
Adenosine Triphosphatases* ; Electrophoresis ; Electrophoresis, Polyacrylamide Gel ; Heart Rate ; Heart* ; Ischemia ; Ischemic Preconditioning* ; Isoenzymes ; Myocytes, Cardiac ; Myosin Heavy Chains ; Myosins* ; Phosphorus ; Reperfusion ; Sodium ; Ventricular Function, Left*

It has been reported that repetitive brief periods of ischemia and reperfusion (ischemic preconditioning, IP) cause a significant reduction in the extent of myocardial necrosis or in the incidence of reperfusion arrhythmias in rat heart. However, recent reports have stated that IP effect is diminished or lost in the canine or bovine heart if ischemia (mostly regional) is sustained for 40 min or longer. The main objective of this study is to assess whether IP provides myocardial protection in prolonged sustained ischemia under the condition of global ischemia in isolated rabbit heart. The hearts were subjected to 10-60 min sustained ischemia (SI) followed by 60 min reperfusion with (IP heart) or without IP (ISCH heart). IP was induced by 4 cycles of 5 min global ischemia and 5 min reperfusion. Left ventricular function (LVF), extent of infarction (EI) and ultrastructural changes were examined. As a whole, the LVF began to recover on reperfusion but there was no significant difference in the functional parameters. However, extracellular Ca2+ concentration was lower in the ISCH hearts (p <0.05) and the EI was significantly different between the hearts which had received 60 min SI (67% in the ISCH versus 32% in the IP heart, p <0.01). Ultrastructural changes were homogeneous in the ISCH hearts and became irreversible in accordance with increase of the duration of ischemia, while these changes were heterogeneous and restricted in the IP heart. These results suggest that IP does not attenuate the postischemic dysfunction in prolonged ischemia but it can provide an infarct size-limiting effect and delay ultrastructural changes. This cardioprotective effect may be related to calcium homeostasis.
Animal ; Arrhythmia/prevention & control ; Calcium/metabolism ; Extracellular Space/metabolism ; Female ; Heart/physiology ; Hydrogen-Ion Concentration ; Male ; Myocardial Infarction/prevention & control ; Myocardial Ischemia/metabolism/*physiopathology ; Myocardial Reperfusion Injury/*prevention & control ; Myocardium/metabolism/pathology/ultrastructure ; Necrosis ; Rabbits ; Support, Non-U.S. Gov't ; Time Factors ; Ventricular Function, Left/physiology