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PubMed2001

Effectiveness of porto-intracaval shunt to reduce the negative effects of portal and caval clamping in the rabbit.

In performing experimental liver surgery, it is difficult to prolong anhepatic time because the animals do not tolerate prolonged portal and caval clamping. To counteract prolonged venous stasis, the authors previously developed a simple porto-intracaval shunt. The shunt consists of a self-constructed inverted Y silicone tube. The effectiveness of this shunt was studied comparing two groups of 10 rabbits with shunt (S) versus those with clamped portal and inferior caval vein (C). In the group of rabbits that underwent porto-intracaval shunt, the results concerning intraoperative mortality, intraoperative increase in distal portal vein pressure, and incidence of the histologic signs of gut damage were clearly improved. The proposed porto-intracaval shunt was therefore effective in reducing some principal negative effects of portal and caval clamping. This type of porto-intracaval shunt can be therefore useful allowing improvement of experimental models concerning liver surgery in little animals.

Animals↗
PubMedDate not supplied

Use of a tilted double clamp in microvascular anastomosis.

A tilted or angulated double microvascular clamp is described that, when applied to the vessels to be anastomosed, presents the vessel ends angulated upward facing the surgeon, so that the initial suturing of the posterior wall can be completed without rotating the vessel. Such a clamp is valuable in clinical situations where space is limited and where conventional double clamps cannot be rotated in the usual fashion.

Constriction↗
PubMed1990

Assessment of microvascular clamping injury of rat aorta by histochemical examination of oxidoreductases.

To assess clamping injury to the rat abdominal aorta, the activities of two oxidoreductases, lactate dehydrogenase (LD) and succinate dehydrogenase (SD), were estimated histochemically at various points in time after clamping the aorta for 30 min (group A) or 60 min (group B) with two kinds of microvascular clips. Areas of depressed LD and SD activity were found 3 days after surgery in group A and 1 day and 3 days after surgery in group B. The random, intermittent occurrence of the changes in enzyme activity within the time groups pointed to a mild trauma induced by the clips. At 1 week and thereafter, no changes in enzyme activities were observed in the aortic wall. Two different patterns of depressed enzyme activity were observed: One was a narrow zone without activity throughout the medial layer, and the other consisted of a broader area of depressed activity in the outer parts of the media. Occluding the aorta with a mosquito clamp led to tissue destruction and loss of enzyme activity in the injured area. This study shows that enzyme histochemistry provides nonquantitative methods to detect and localize microvascular injury and that the injury caused by the microvascular clips was mild and reversible.

Animals↗
PubMed1997

Effect of nitroglycerin and cerebrospinal fluid drainage on spinal cord perfusion pressure and paraplegia during aortic cross-clamping.

When sodium nitroprusside (SNP) is used to control proximal blood pressure (Px-BP) during cross-clamping (AXC) of the thoracic aorta, it decreases spinal cord perfusion pressure (SCPP) by reducing distal aortic pressure (Ds-BP) and increasing cerebrospinal fluid pressure (CSFP). The decrease cannot be reversed by CSF drainage (CSFD) because such drainage is limited by a reduction in compliance of the spinal canal. Nitroglycerin can also be used to control Px-BP, but its effect on CSF dynamics has not previously been investigated. In the present study we have compared the effects of NTG alone and in combination with CSFD, with SNP and CSFD. Each group (Gp) of six dogs was treated with SNP + CSFD (Gp 1), NTG alone (Gp 2), and NTG + CSFD (Gp 3). Left carotid and right femoral arteries were catheterized to monitor Px-BP and Ds-BP, respectively. CSFP was monitored and CSF was drained through a spinal needle placed in the cisterna cerebellomedullaris. The thoracic aorta was cross-clamped via a left thoracotomy for 60 min. Data were acquired at baseline, during aortic occlusion, and 24 hr after surgery. There were no significant differences in any measurements among the three groups before AXC; after AXC, Px-BP was maintained between 85 and 95 mm Hg in all groups. Ds-BP was significantly lower in Gp 1 than Gp 2 and 3 (7 +/- 2 mm Hg vs. 13 +/- 3 mm Hg and 17 +/- 2 mm Hg, respectively P < 0.05). CSFP did not differ between Gp 1 and 2 (10 +/- 3 mm Hg vs. 9 +/- 1 mm Hg, P > 0.05). CSFD effectively kept CSFP at zero values in Gp 3 during AXC, but not in Gp 1. SCPP was significantly higher in Gp 3 than in Gp 1 and 2 (17 +/- 2 mm Hg vs -3 +/- 4 mm Hg and 4 +/- 1 mm Hg, respectively, P < 0.05). All animals in Gp 1 and 2 suffered paraplegia, as opposed to none in Gp 3. NTG causes paraplegia by decreasing SCPP. When used in conjunction with CSFD, it controls Px-BP without causing paraplegia. CSFD cannot counteract the negative effects of SNP on SCPP; therefore, SNP contributes to postoperative paraplegia. The effects of NTG on cerebrospinal fluid dynamics are different from those of SNP. We caution surgeons against the use of NTG without CSFD during aortic cross-clamping.

Animals↗
PubMed1983

Effect of coenzyme Q10 on hypertrophied ischemic myocardium during aortic cross clamping for 2 hr, from the aspect of energy metabolism.

In order to perform intracardiac repair safely during aortic cross clamping, we designed this study to evaluate the protective effect of coenzyme Q10 (CoQ10) on hypertrophied ischemic myocardium from the aspect of energy metabolism. Six to nine months preceding the study, aortic bandings were carried out on 14 puppies to produce left ventricular hypertrophy (LVH). These dogs with LVH were then subjected to total cardiopulmonary bypass and were evenly divided into control and CoQ10-treated groups (10 mg/kg of intravenous administration plus 1 mg/kg per hr of intracoronary injection). Myocardial ischemia was induced by aortic cross clamping for 2 hr under moderate systemic hypothermia. The results indicated that the administration of CoQ10 had a protective effect on hypertrophied ischemic myocardium, since depletion of high-energy phosphate (HEP) was uniformly prevented, and accumulation of lactate was simultaneously decreased during the 2 hr of aortic cross clamping. On the other hand, there were marked exhaustion of HEP and rapid increase in lactate following the 2 hr of ischemia in the control group, these being much more predominant in the subendocardial layer.

Adenosine Triphosphate↗
PubMed1989

Electrophysiological actions of A23187 and X-537A in spontaneously beating and in voltage-clamped rabbit sino-atrial node preparations.

Electrophysiological effects of calcium ionophores, A23187 and X-537A, on spontaneously beating and voltage-clamped rabbit sino-atrial node preparations were examined, using the voltage-clamp technique with two microelectrodes. (1) A23187 (administered cumulatively) increased the cycle length significantly at 3 x 10(-6) and 10(-5) mol/l, and X-537 only at 10(-5) mol/l. Other action potential parameters were unaffected in the presence of these concentrations of either agent. At 2 x 10(-5) mol/l, either agent prolonged the cycle length significantly, but increased the amplitude and the duration of the action potentials and the maximum diastolic potential not to any significant extent. Both X-537A and A23187, at 2 x 10(-5)mol/l, induced a dysrhythmia, which in the former was probably due to delayed afterdepolarizations. (2) In voltage-clamped sino-atrial node cells, the holding current was shifted outwardly, to a greater extent in the presence of X-537A than A23187 at the same concentration (2 x 10(-5) mol/l). The ionophores initially increased the slow inward current and then decreased it. The steady outward current was inhibited, and its activation curve was shifted to a more negative voltage range. X-537A caused a transient inward current and an inward tail current on repolarization to the holding potential. (3) At concentrations of 10 and 18 mmol/l [Ca2+]o or in the presence of isoprenaline 10(-7) mol/l, these ionophores induced a more severe dysrhythmia. Conversely in the nominal absence of [Ca2+]o the regular rhythm was resumed.(ABSTRACT TRUNCATED AT 250 WORDS)

Action Potentials↗
PubMed1994

Configuration of light responses in isolated retinal rods. A patch-clamp study.

The whole-cell patch-clamp technique was employed to investigate the light responses of single retinal rods of the frog (Rana esculenta and R. temporaria). In the majority of experiments, completely isolated cells were studied. Coupling with neighboring cells gave rise to a more complex response configuration. Responses were recorded under voltage-clamp and under current-clamp conditions. Stimulus response curves were measured in experiments with local stimuli illuminating only parts of the outer segment. Metabolic factors such as cGMP, GTP and ATP were also tested and were found to have specific and different influences on the response configurations. When the recording pipette was filled with an intracellular medium devoid of nucleotides, a retardation in the recovery of the light responses was observed during the course of an experiment. Addition of 1 mM ATP to the pipette medium prevented the larger part of the retardation, while 1 mM GTP accelerated the response recovery at the beginning of an experiment but did not prevent a subsequent retardation. Micromolar concentrations of cGMP were sufficient to elicit both a depolarization of the photoreceptor membrane and an increase in the response duration. These results show that, in single photoreceptors, the configuration of light responses not only depends on the stimulus parameters but also on those properties of the cells that are directly controlled by their nucleotide metabolism.

Adenosine Triphosphate↗
PubMedDate not supplied

Measurement of whole-cell calcium current in voltage-clamped vascular muscle cells.

The direct measurement of transmembrane calcium current in single vascular muscle cells has been accomplished recently using the whole-cell voltage-clamp technique. The small size of the vascular muscle cell and the proportionately smaller magnitude of its inward calcium current necessitate refined instrumentation, but also make the vascular muscle cell an ideal candidate for whole-cell voltage-clamp recording. Calcium current in vascular muscle cells appears to have some, but not all, characteristics in common with calcium currents similarly isolated in neuronal and cardiac cells, including voltage-dependent activation and steady-state inactivation of calcium current, the presence of two current types, and sensitivity to inorganic and organic calcium channel modulating drugs. Future voltage-clamp analysis of calcium currents in vascular muscle is needed to further our understanding of the control of the calcium channels in physiological and pathophysiological states.

Animals↗
PubMed1989

Curvature-electric effects in artificial and natural membranes studied using patch-clamp techniques.

Methods for applying sound pressure to membrane patches formed at the tips of patch-clamp pipettes have been developed. Artificial membrane patches were formed from diphytanoyl phosphatidylcholine using a pipette dipping technique. Natural membrane patches were excised (inside-out mode) from collagenase-treated locust muscle membrane. Curvature-electric signals were registered under both voltage clamp and current clamp conditions. The phenomenon of flexoelectricity in membranes has previously been attributed to curvature-induced polarization originating from the liquid crystalline properties of membranes. The estimated magnitude (2 x 10(-18) C) of the flexoelectric coefficient of the artificial lipid bilayers is consistent with previous findings while that of the muscle membrane was in certain cases several times larger. The present study is the first to report on flexoelectricity in a natural membrane and raises the question of the biological significance of this phenomenon.

Animals↗
PubMed1981

The metabolic response to hyperglycaemic clamping in insulin-dependent diabetes.

The metabolic and hormonal effects of stable hyperglycaemia (10-12 mmol/l) have been examined in five insulin-dependent diabetics and compared with the results of 8 h (1200 to 2000 h) normoglycaemic (5-6 mmol/l) clamping. Glucose levels were maintained using a glucose controlled insulin infusion system. Mean blood lactate, pyruvate, total ketone bodies, glycerol and plasma non-esterified fatty acids were similar during the period of stable glycaemia at the two glucose levels. In contrast mean blood alanine was markedly elevated during hyperglycaemic clamping (0.384 +/- 0.008 vs 0.298 +/- 0.021 mmol/l) and 3-hydroxybutyrate was slightly decreased (0.068 +/- 0.007 vs 0.084 +/- 0.008 mmol/l). Plasma glucagon levels were raised during hyperglycaemic clamping and growth hormone slightly decreased. There was a close positive correlation between mean blood alanine and mean blood glucose (r = 0.79, p less than 0.01), and a negative correlation of alanine with the amount of insulin infused (r = -0.72, p less than 0.01). It is suggested that the raised alanine results from increased peripheral glucose utilisation. In general a short period of stable hyperglycaemia is not associated with a worsening of metabolic abnormalities in insulin-dependent diabetic subjects.

Adult↗
PubMed1994

Voltage-dependent Ca2+ influx in the epithelial cell line HT29: simultaneous use of intracellular Ca2+ measurements and nystatin perforated patch-clamp technique.

Indirect evidence has accumulated indicating a voltage dependence of the agonist-stimulated Ca2+ influx into epithelial cells. Manoeuvres expected to depolarise the membrane voltage during agonist stimulation resulted in: (1) a decrease of the sustained phase of the adenosine triphosphate (ATP, 10(-5) mol/l)-induced intracellular Ca2+ transient, (2) a reduced fura-2 Mn(2+)-quenching rate, and (3) prevention of the refilling of the agonist-sensitive store. To quantify the change in intracellular Ca2+ as a function of membrane voltage, we measured simultaneously the intracellular Ca2+ activity ([Ca2+]i) with fura-2 and the electrical properties using the nystatin perforated patch-clamp technique in single HT29 cells. Ca2+ influx was either stimulated by ATP (10(-5) mol/l) or thapsigargin (TG, 10(-8) mol/l). After [Ca2+]i reached the sustained plateau phase we clamped the membrane voltage in steps of 10 mV in either direction. A stepwise depolarisation resulted in a stepwise reduction of [Ca2+]i. Similarly a stepwise hyperpolarisation resulted in a stepwise increase of [Ca2+]i (ATP: 27.5 +/- 10 nmol/l per 10 mV, n = 6; TG: 19 +/- 7.9 nmol/l per 10 mV, n = 12). The summarised data show a linear relationship between the delta fluorescence ratio 340/380 nm change and the applied holding voltage. In unstimulated cells the same voltage-clamp protocol did not change [Ca2+]i (n = 9). Under extracellular Ca(2+)-free conditions [Ca2+]i remained unaltered when changing the membrane voltage. These data provide direct evidence that the Ca2+ influx in epithelial cells is membrane voltage dependent. Our data indicate that small changes in membrane voltage lead to substantial changes in [Ca2+]i.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1989

Calcium currents in single SA nodal cells of the rabbit heart studied with action potential clamp.

With a new method called "Action Potential-Clamp" (APC) we studied in single SA nodal cells the contribution of both transient and long lasting calcium currents (L-type and T-type) to the action potential. Action potentials were recorded by a computer and a representative cycle was subsequently repetitively replayed to the same cell under voltage clamp. Blockade of the L-type calcium current (D600) or T-type calcium current (nickel) revealed the quantitative and time related contributions of these currents to the action potential, since the blocked current is compensated by the clamp amplifier.

Action Potentials↗
PubMed1987

On the relationship between V max of slow responses and Ca-current availability in whole-cell clamped guinea pig heart cells.

The relationship between Ca current availability and maximum rate of rise (V max) of slow responses was determined in the same single guinea pig ventricular heart cell under voltage and current clamp conditions (whole-cell clamp technique). The results are as follows. (1) Cell capacitance measured in 32 cells from the current response to a fast ramp voltage-clamp pulse (119.6 +/- 4.6 pF, mean +/- SE) or from Vmax values at a holding potential of -50 or -40 mV (118.6 +/- 5.3 pF) are identical. (2) In control conditions ([Ca]o 1.8, [K]o 4 and [Cs]i 140 mM), voltage-dependence of steady-state inactivation of Ca current (ICa) or Vmax are similar up to -35 mV. However, Vmax significantly (P less than 0.005) underestimates ICa availability at more positive potentials. At -30 mV, ICa and Vmax amplitudes represent respectively 35.6 and 22.4% (n = 14) of their maximum value. (3) In the presence of 50 nM isoprenaline, Vmax and the underlying ICa are respectively increased by 79.2 +/- 13.8% and 71.2 +/- 13.8% (n = 15). No statistically significant deviation from linearity is then observed. (4) When Vmax amplitude is expressed as a function of ICa density, an almost linear relationship is observed for Vmax values between 0 and 25 V/s. Vmax is then best described by the equation: Vmax (V/s) = 1.043 ICa (pA/pF) -0.514 (46 cells). (5) We conclude that, under conditions that minimize outward currents, Vmax of slow responses accurately measures ICa amplitude, except when ICa is decreased to less than 40% of its maximum control amplitude (i.e., below 4 pA/pF). At that point, Vmax underestimates ICa.

Action Potentials↗
PubMed1984

An improved loose patch voltage clamp method using concentric pipettes.

A method for noninvasive voltage-clamp recording from large cells is described. A firepolished pipette having two concentric barrels is pushed against the cell membrane, thereby electrically isolating a circular patch subdivided into an inner and an annular outer region. Both regions are held isopotential, but current is collected from the inner region only. The method electrically simulates a high resistance seal between pipette and cell membrane, allowing accurate and rapid voltage-clamp recording under conditions where the seal resistances actually obtained are low (near 1 M omega). This is useful in applications where one wishes to avoid enzymatic treatment. We provide details of electrode construction and voltage-clamp electronics, and present results obtained from frog skeletal muscle and leech neurons. For sodium channels of frog muscle, extensive data were previously obtained with other methods. There is good agreement between the earlier results and the measurements presented here.

Animals↗
PubMed1978

Transient tension responses of voltage-clamped frog atrial muscle related to sudden changes in external Ca or Na.

Frog atrial muscle strips were placed in a double sucrose gap chamber and perfusion of the central node was arranged as to allow rapid changes of external Ca or Na concentration during long-lasting (15 s) depolarizing clamps. When the superfusing fluid was suddenly switched, the intercellular space inside the fibre bundle equilibrated with a time constant in the order of 1 s. A fast change of perfusate during clamp was followed by a delayed change of tonic (sustained) contraction. When [Ca]0 was increased from 0.25 to 4 mM, tension rose in a sigmoid manner and the level reached at the end of the clamp was almost identical with the steady control in 4 mM-Ca-Ringer. A similar tension increase occurred upon a reduction of [Na]0 from 100 to 25% of normal. At a given depolarization time course and height of the tension responses followed the ratio of [Ca]0/[Na]20. Transient tension responses are interpreted in terms of a sudden perturbation of a transmembrane Ca-Na exchange system leading to a depression of Ca pumping activity.

Animals↗
PubMed1977

Depletion and accumulation of potassium in the extracellular clefts of cardiac Purkinje fibers during voltage clamp hyperpolarization and depolarization.

1. Voltage clamp hyperpolarization and depolarization elicited current records consistent with depletion and accumulation, respectively, of potassium in the extracellular clefts of cardiac Purkinje fibers. Hyperpolarization was shown to shift the reversal potential for the pacemaker current, ik2, a measure of Ek, to more negative potentials. Upon depolarization, a slowly increasing outward current was observed. Analysis of the tail currents elicited by hyperpolarization revealed that a time-dependent change in gx could not explain the time-dependent outward current. However, the tail currents were consistent with a shift of Ek to more positive potentials during the depolarization. 2. Alteration in potassium driving force over time results in a time-dependent ik1 even though the underlying conductance is time-independent [29]. This time-dependent current may contribute to the currents usually identified as ik2 and ix. 3. The potential at which ik2 reverses direction is altered by the clamp program used to elicit it and is obscured by the superimposition of a time-dependent current due to depletion. 4. Records consistent with the extracellular cleft potassium concentration being less than that of the bulk phase in the quiescent fiber were obtained. However, an unequivocal interpretation of these current reocrds could not be made. 5. These results suggest that conclusions based on the assumption that potassium driving force remains constant during a voltage clamp pulse may be in error. Thus, time-dependent currents cannot be assumed to result solely from time-dependent conductance changes.

Animals↗
PubMed1982

Computed tomography stereotactic head clamp.

A new stereotactic head clamp is described. The clamp is applied in the plane of the computed tomographic scan, and enables one to probe the target in te plane of the scan by obtaining measurements directly from the scanner. No modification of the scanner is required, and the clamp can be used within the scanner.

Adenoma, Chromophobe↗
PubMed1988

Voltage- and time dependence of apical membrane conductance during current clamp in Necturus gallbladder epithelium.

The effects of short (1 sec) and long (1 min) transepithelial current clamps on membrane voltages and resistances of Necturus gallbladder were investigated. Transepithelial and cell membrane current-voltage relationships determined from 1-sec clamps revealed that: a) depolarization of the apical membrane voltage (Vmc) results in a marked decrease in apical membrane fractional resistance (fRa), whereas hyperpolarization of Vmc results in either no change in fRa or a small increase, and b) the voltage-dependent changes in fRa are essentially complete within 500 msec. Exposure of the tissue to 5 mM TEA+ on the mucosal side caused no significant change in baseline Vmc (-69 +/- 2 mV) and yet virtually abolished the voltage dependence of fRa. A possible interpretation of these results is that two types of K+ channels exist in the apical membrane, with different voltage dependencies and TEA+ sensitivities. Acidification or Ba2+ addition to the mucosal solution also reduced the voltage-dependent changes in fRa. The time courses of the changes in fRa and in the cable properties of the epithelium were assessed during 1-min transepithelial current clamps (+/- 200 microA/cm2). No secondary change in fRa was observed with mucosa-to-serosa currents, but a slow TEA+-sensitive decrease in fRa (half-time of seconds) was evident with serosa-to-mucosa currents. Cable analysis experiments demonstrated that the initial (less than 500 msec) voltage-dependent decrease in fRa is due to a fall in apical membrane resistance. The later decrease in fRa is due to changes in both cell membrane resistances attributable to the increase in transcellular current flow resulting from a fall in paracellular conductance. The voltage dependence of the apical membrane conductance is a more significant problem in estimating fRa than the current-induced effects on the lateral intercellular spaces. In principle, TEA+ can be used to prevent the nonlinear behavior of Ra during measurements of the voltage divider or membrane resistance ratio.

Animals↗