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PubMed1975

Axon voltage-clamp simulations. II. Double sucrose-gap method.

This is the second in a series of four papers on the simulation of the voltage clamp of cylindrical excitable cells. In this paper we evaluate the double sucrose-gap voltage-clamp technique for the squid and lobster giant axons. Using the Crank-Nicolson method of solution of the cable equations and differential equations representing the voltage clamp circuit we studied the effect of length of the sucrose gap "node" on the voltage profile along an excitable cell during a simulated voltage clamp. The voltage gradients along the region of the cell within the node produce "notches" in the current recording as well as changes in the magnitude of the sodium and potassium current for a given voltage step. Our results show that good voltage clamp control requires node lengths less than one-half the axon diameter.

Action Potentials↗
PubMed1975

Axon voltage-clamp simulations. III. Postsynaptic region.

This is the third in a series of four papers in which we present the numerical simulations of the application of the voltage clamp technique to excitable cells. In this paper we discuss the problem of voltage clamping a region of a cylindrical cell using microelectrodes for current injection and voltage recording. A recently developed technique (Llinás et al., 1974) of internal application of oil drops to electrically insulate a short length of the postsynaptic region of the squid giant synapse is evaluated by simulation of the voltage clamp of an excitable cylindrical cell of finite length with variable placement of the current and voltage electrodes. Our results show that ENa can be determined quite accurately with feasible oil gap lengths but that the determination of the reversal potential for the synaptic conductance, ES, can be considerably in error. The error in the determination of ES dependp, and especially the membrane resistance at the time the synaptic conductance occurs. It is shown that the application of tetraethylammonium chloride to block the active potassium conductance very significantly reduces the error in the determination of ES. In addition we discuss the effects of cable length and electrode position on the apparent amplitude and time course of the syn aptic conductance change. These results are particularly relevant to the application of the voltage clamp technique to cells with nonsomatic synapses. The method of simulation presented here provides a tool for evaluation of voltage clamp analysis of synaptic transmission for any cell with known membrane parameters and geometry.

Action Potentials↗
PubMed1987

Theory of the kinetic analysis of patch-clamp data.

This paper describes a theory of the kinetic analysis of patch-clamp data. We assume that channel gating is a Markov process that can be described by a model consisting of n kinetic states and n(n - 1) rate constants at each voltage, and that patch-clamp data describe the occupancy of x different conductance levels over time. In general, all the kinetic information in a set of patch-clamp data is found in either two-dimensional dwell time histograms describing the frequency of observation of sequential dwell times of durations tau 1 and tau 2 (Fredkin, D. R., M. Montal, and J. A. Rice, 1985, Proceedings of the Berkeley Conference in Honor of Jerzy Neyman and Jack Kiefer, vol. 1, 269-289) or in three-point joint probability functions describing the probability that a channel is in a given conductance at time t, and at time t + tau 1, and at time t + tau 1 + tau 2. For the special case of channels with a single open state plus multiple closed states, one-dimensional analyses provide all of the kinetic information. Stationary patch-clamp data have information that can be used to determine H rate constants, where H = n(n - 1) - G and G is the number of intraconductance rate constants. Thus, to calculate H rate constants, G rate constants must be fixed. In general there are multiple sets of G rate constants that can be fixed to allow the calculation of H rate constants although not every set of G rate constants will work. Arbitrary assignment of the G intraconductance rate constants equal to zero always provides a solution and the calculation of H rate constants. Nonstationary patch-clamp data have information for the determination of H rate constants at a reference voltage plus n(n - 1) rate constants at all test voltages. Thus, nonstationary data have extra information about the voltage dependencies of rate constants that can be used to rule out kinetic models that cannot be disqualified on the basis of stationary data.

Electric Conductivity↗
PubMed1989

Ionic diffusion in voltage-clamped isolated cardiac myocytes. Implications for Na,K-pump studies.

The whole-cell voltage-clamp technique employing electrolyte-filled micro-pipette suction electrodes is widely used to investigate questions requiring an electrophysiological approach. With this technique, the ionic composition of the cytosol is assumed to be strongly influenced (as result of diffusion) by the ionic composition of the solution contained in the electrode. If this assumption is valid for isolated cardiac myocytes, the technique would be particularly powerful for studying the dependence of their Na,K-pump on the intracellular [Na+]. However, the relationship between the concentrations of ions in the solution filling the electrode and those in the cytosol has not been established. The relationship was investigated to determine in particular whether the [Na+] at the intracellular cation ligand binding sites for the Na-pump ([ Na+]ps) can be set and clamped by [Na+] in the pipette electrode ([ Na+]pip). If [Na+]pip can set and clamp [Na+]ps, this would provide a means for defining the dependence of the Na,K-pump on intracellular [Na+]. The relationship between [Na+]pip and [Na+]ps was analyzed using two approaches. First, a mathematical model of three-dimensional ionic diffusion within a whole-cell patch-clamped myocyte was developed and the effects of experimental parameters on mean [Na+]ps were investigated. When typical experimental values were simulated, the time course to achieve steady state mean [Na+]ps was found to be most sensitive to variations in electrode pore size, cell length and the Na+ pumping rate, but at steady state, mean [Na+]ps varies from [Na+]pip by 5% or less depending on pump rate. Second, to provide experimental support for the validity of the simulations, isolated ventricular myocytes were voltage-clamped and the reversal potential for the Na current was determined in order to estimate steady state intracellular [Na+]. The results of the mathematical and experimental analyses suggest that steady state [Na+]ps can be regulated by the [Na+] in suction pipette electrodes. These findings, while also having a broader significance, indicate for isolated cardiac myocytes that whole-cell suction micro-electrodes can provide a means to assess the dependence of the Na,K-pump on [Na+]ps.

Algorithms↗
PubMed1999

Supraceliac aortic clamping during the anhepatic phase of experimental orthotopic liver transplantation.

BACKGROUND: The pig tolerates simultaneous clamping of the liver pedicle and inferior vena cava poorly, so venovenous bypass has to be used during the anhepatic phase of experimental orthotopic liver transplantation (OLT). The aim of this work is to assess whether clamping of the supracoeliac aorta during the anhepatic phase (AP) of experimental OLT in pigs allows transplantation in stable hemodynamic conditions. METHODS: Fourteen pigs (weight, 16 to 18 kg) received whole liver grafts from 14 age-matched donors and were subsequently divided into two groups: group I, OLT without venovenous bypass during the AP, group II, OLT with supracoeliac aortic clamping during the AP. Variables analyzed were cardiac output (CO) and related variables, mean systemic arterial pressure (MAP), mixed venous oxygen saturation (SvO2), hepatic artery and portal vein blood flow, systemic and hepatic O2 supply and uptake (SDO2, SVO2, HDO2, HVO2, respectively), liver enzymes, glucose, creatinine, and electrolytes. RESULTS: In group I, CO, MAP, and SvO2, decreased during the AP (anhepatic) in comparison with baseline (preanhepatic) values (CO, 3.60+/-0.74, preanhepatic, v. 1.21+/-0.25 L x min(-1), anhepatic; P<.05. MAP, 97+/-12, preanhepatic, v. 43+/-17 mm Hg, anhepatic; P<.05. SvO2, 91.6+/-5.6, preanhepatic v. 70.0+/-12.5%, anhepatic; P<.05), and SDO2/SVO2 increased by 16% (preanhepatic) to 33% (anhepatic; P<.05). In group II, CO decreased during the anhepatic phase by only 21% (3.82+/-0.81, preanhepatic, v. 3.07+/-0.99 L x min(-1), anhepatic; not significant), the MAP increased significantly (100+/-8, preanhepatic, v. 135+/-4 mm Hg, anhepatic; P<.05), and SVO2, SDO2, SVO2, and SDO2/SVO2 remained unchanged. After revascularization, none of these variables differed significantly between groups, and levels of liver enzymes, glucose, creatinine, urea, and electrolytes were similar in both groups, both before and aftertransplantation. CONCLUSIONS: Experimental OLT can be carried out in pigs without venovenous bypass, but it leads to severe hemodynamic disturbances. Clamping of the supraceliac artery during the AP is well tolerated and results in excellent hemodynamic stability, so it may prove to be a useful technique in liver transplantation in animals, such as dogs or pigs, that do not tolerate simultaneous clamping of the liver pedicle and inferior vena cava as well as human beings.

Animals↗
PubMed1998

The Gupta-Frank clamp for salpingo-oophorectomy at vaginal hysterectomy.

BACKGROUND: When performing a vaginal hysterectomy, removal of the tubes and ovaries can be either necessary or desirable. We describe our experience with a new instrument called the Gupta-Frank clamp for performing vaginal salpingo-oophorectomy at the time of vaginal hysterectomy. INSTRUMENT: The stainless steel Gupta-Frank clamp is 260 mm long, and its shanks are curved to avoid impedance from the vaginal walls. The 70-mm jaws have a smooth curve, which allows both the round and infundibulopelvic ligaments to be incorporated into a single clamp bite. Each jaw has three serrated grooves, which interlock with the corresponding jaw grooves to give a 6-mm-wide atraumatic nonslip area. EXPERIENCE: We have used this instrument in 18 women undergoing bilateral salpingo-oophorectomy at the time of vaginal hysterectomy. A single clamp bite on each side was sufficient to accomplish salpingo-oophorectomy in all cases. There were no major complications. CONCLUSION: Salpingo-oophorectomy performed vaginally at the time of vaginal hysterectomy with the Gupta-Frank clamp is feasible and is accomplished easily and safely.

Equipment Design↗
PubMed2000

Bone flap fixation with titanium clamps: a new technique.

BACKGROUND: Craniotomy bone flaps are replaced for both cosmetic and protective purposes. The purpose of this study was to determine the facility, strength, timing, and cost effectiveness of an alternative system for securing bone flaps. The system consists of titanium discs that clamp the bone flap to the skull edge. METHODS AND RESULTS: Ten cadaver craniotomy flaps each were reattached with either 24-gauge wire, miniplates, or titanium clamps. The titanium clamp system required significantly less time to fix than either wire or miniplates. The clamps were stronger than wire and cost less than miniplates. CONCLUSION: The titanium clamp system is a reasonable alternative to present craniotomy fixation methods with respect to ease of use, time consumption, strength, and relative cost.

Craniotomy↗
PubMed1999

Building a replisome from interacting pieces: sliding clamp complexed to a peptide from DNA polymerase and a polymerase editing complex.

We have solved the crystal structures of the bacteriophage RB69 sliding clamp, its complex with a peptide essential for DNA polymerase interactions, and the DNA polymerase complexed with primer-template DNA. The editing complex structure shows a partially melted duplex DNA exiting from the exonuclease domain at an unexpected angle and significant changes in the protein structure. The clamp complex shows the C-terminal 11 residues of polymerase bound in a hydrophobic pocket, and it allows docking of the editing and clamp structures together. The peptide binds to the sliding clamp at a position identical to that of a replication inhibitor peptide bound to PCNA, suggesting that the replication inhibitor protein p21CIP1 functions by competing with eukaryotic polymerases for the same binding pocket on the clamp.

Amino Acid Sequence↗
PubMed1994

An explanation for lagging strand replication: polymerase hopping among DNA sliding clamps.

The replicase of E. coli, DNA polymerase III holoenzyme, is tightly fastened to DNA by its ring-shaped beta sliding clamp. However, despite being clamped to DNA, the polymerase must rapidly cycle on and off DNA to synthesize thousands of Okazaki fragments on the lagging strand. This study shows that DNA polymerase III holoenzyme cycles from one DNA to another by a novel mechanism of partial disassembly of its multisubunit structure and then reassembly. Upon completing a template, the polymerase disengages from its beta clamp, hops off DNA, and reassociates with another beta clamp at a new primed site. The original beta clamp is left on DNA and may be harnessed by other machineries to coordinate their action with chromosome replication.

DNA↗
PubMed1989

[A single-use vascular clamp. An experimental and clinical study].

Twelve rabbit femoral arteries were clamped for one hour with a TKS2 clamp in order to confirm the atraumatic nature of the Tamai disposable clamp. After 15 or 30 minutes of revascularisation, the arteries were excised and studied by light microscopy and scanning electron microscopy. Temporary dilatation of the artery was observed after removal of the clamp, as the patency test was always positive. Light microscopy revealed parietal flattening without any cellular disorganisation and scanning electron microscopy revealed flattening of the endothelial cells without any tearing. On the basis of these favourable results, combined with the ease of use, we now use the Tamai clamp daily.

Animals↗
PubMed1987

Disseminated intravascular coagulation as a result of supraceliac clamping: implications for thoracoabdominal aneurysm repair.

Massive coagulopathy and bleeding continues to play a major role in the operative mortality and perioperative multi-system failure of patients requiring elective thoracoabdominal aneurysm repair. It was the purpose of this study to determine the coagulation defect that occurs with supraceliac aortic clamping and the effects of increasing aortic cross-clamp time (AXCT) on the coagulation system and its recovery. Through a standard thoracoabdominal incision, 16 mongrel dogs had their aortas cross-clamped simultaneously just above the diaphragm and at the aortic bifurcation. Animals were divided into four groups of four animals each; sham operation, 30 minute AXCT, 60 minute AXCT, and 90 minute AXCT. Central venous blood was sampled prior to aortic cross clamping (AXC), during AXC and 1 hour, 2 hours, 5 hours, 7 hours, 12 hours, and 24 hours after the clamp was removed. All samples were assayed for platelets, fibrinogen, fibrin split products, prothrombin time (PT) and partial thromboplastin time (PTT). Platelets and fibrinogen decreased as PT and PTT increased with increasing AXCT consistent with disseminated intravascular coagulation (DIC) (P less than .001). Fibrin split products were positive in the 90 minute AXCT group only. The drop in platelets was greater for increasing AXCT and continued to fall in the 30, 60 and 90 minute AXCT groups at 24 hours (p less than .001). Fibrinogen dropped to the lowest levels between two and twelve hours after AXC and returned to normal at twenty-four hours in the 60 and 90 minute AXCT groups (p less than .05).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Immediate effects of carotid clamp release on middle cerebral artery blood flow velocity during carotid endarterectomy.

Transcranial Doppler ultrasound was employed in 44 patients undergoing carotid endarterectomy in order to identify factors associated with the greatest increases in middle cerebral artery blood flow velocity (MCAV) immediately after carotid clamp release and restoration of flow. Previous reports have suggested that such increases might reflect post-ischemic hyperaemia. Overall, the median increase in MCAV on restoration of flow [58 cm/s (95% Cl 44-68)] was greater than the median decrease in MCAV after initial carotid clamping [36 cm/s (95% Cl 26-43), p < 0.0001]. However, the immediate increase in MCAV tended to be transient and, in one patient, may have represented a hyperaemic response to a haemodynamically induced intraoperative neurological deficit. There was no association between the magnitude of MCAV increase and clinical presentation, degree of carotid stenosis, computed tomography scan findings, type of anaesthesia, nor the presence or absence of impaired cerebral vascular reserve, nor the occurrence of intraoperative air embolisation. The greatest increases in MCAV on clamp release were observed in patients with the greatest decreases in MCAV at clamping and those with the lowest internal carotid artery stump pressures. The most likely explanation for the findings is that they represent a transient hyperaemic response to carotid occlusion. It remains unclear whether this phenomenon is mediated by some degree of ischaemic injury or simply by the effect of a sudden surge of blood through low resistance arterioles that have dilated in order to maintain the collateral circulation during carotid clamping.

Blood Flow Velocity↗
PubMed1993

Neuroendocrine and haemodynamic responses to abdominal aortic cross clamp and release during high-dose opiate-oxygen-isoflurane anaesthesia.

The haemodynamic and neuroendocrine responses and beta adrenoceptor distribution associated with aortic cross clamping and release were quantified in 14 patients undergoing elective abdominal aortic aneurysm surgery using a high-dose opiate-oxygen-isoflurane anaesthetic technique. These changes were correlated with neutrophil beta adrenoceptor distribution. Aortic cross clamp application was associated with increased systemic vascular resistance (SVR), and decreased cardiac index (CI). Left ventricular stroke work index remained constant during the period of cross clamp application and following release. Cross clamp release was associated with increased CI and decreased SVR. Plasma cortisol concentrations did not change during the study period. Plasma catecholamine concentrations, although elevated prior to surgery, remained unchanged during aortic cross clamping and following release. The percentage of internalised beta adrenoceptors was elevated before surgery and was unaffected by surgical intervention. This study suggests that a high-dose fentanyl-oxygen-isoflurane anaesthetic technique in a patient population with high circulating catecholamine levels and downregulation of beta adrenoceptors is associated with cardiovascular stability and attenuated neuroendocrine responses.

Aged↗
PubMed1993

Cerebral haemodynamics during carotid cross-clamping.

Carotid artery cross-clamping ischaemia during carotid endarterectomy (CEA) causes 5-30% of perioperative neurological deficits. This study was performed to identify possible clinical situations at higher risk for carotid cross-clamping ischaemia. 606 consecutive patients underwent CEA and were retrospectively studied; they were grouped according to risk factors, presence of associated vascular diseases, clinical pattern, angiographic and CT scan findings. Stump pressure measurement was provided in all patients, perioperative monitoring during CEA was performed by electroencephalogram (EEG) in 469 (77%) and somatosensorial evoked potentials (SEP) in 137 (23%). Local anaesthesia was used in 88 (14.5%) patients. Ischaemic changes during carotid cross-clamping were registered in 118 patients (19.5%). The incidence of cross-clamping ischaemia was then related to different factors; it affected 5.6% of asymptomatics, 25.4% of patients with fixed stroke and 38.5% of those with stenosis and contralateral occlusion. Angiographic and clinical correlation showed that patients with more severe lesions are mostly affected by clamping ischaemia (up to 55% in those with stroke and stenosis with contralateral occlusion). Age, hypertension and diabetes do not significantly affect incidence of ischaemic changes. Positive CT scan increased this risk; statistical relevance was found in regard to patients with unilateral or bilateral stenosis and in those with transient ischaemic attacks. A higher risk can be expected for subjects with more severe clinical and instrumental findings, even if no patients can be considered completely at risk or risk free. Perioperative monitoring is always mandatory and is of great importance in detecting ischaemic changes and preventing cerebral damage using a temporary intraluminal shunt.

Aged↗
PubMed2003

Nucleotide-induced conformational changes in an isolated Escherichia coli DNA polymerase III clamp loader subunit.

Sliding clamps are loaded onto DNA by ATP-driven clamp loader complexes. The structure of the E. coli clamp loader in a nucleotide-free state has been determined previously. We now report crystal structures of a truncated form of the isolated gamma-ATPase subunit, gamma(1-243), of the E. coli clamp loader, in nucleotide-free and bound forms. The gamma subunit adopts a defined conformation when empty, in which the nucleotide binding site is blocked. The binding of either ATPgammaS or ADP, which are shown to bind with equal affinity to gamma(1-243), induces a change in the relative orientation of the two domains such that nucleotides can be accommodated. This change would break one of the gamma:gamma interfaces seen in the empty clamp loader complex, and may represent one step in the activation process.

Adenosine Triphosphatases↗
PubMed2000

Single-clamp technique for aneurysms of the descending thoracic aorta: report of 132 consecutive cases.

OBJECTIVE: To determine the efficacy of a single-clamp technique in preventing spinal cord ischemia during repair of aneurysms of the descending thoracic aorta. PATIENTS AND METHODS: From January 1989 to May 1999, 132 consecutive patients (91 men and 41 women, aged 31-86 years), with aneurysms of the descending thoracic aorta underwent repair using a single-clamp technique and temporary partial distal exsanguination. The diseased aortic segment was replaced with a Dacron graft. Blood was re-infused from an auto-transfusion device, and the segmental vessels were over-sewn but not implanted into the graft. RESULTS: The average aortic cross-clamp time was 26.4 min (range, 11-67 min) for the overall group and 37.4 min for patients who had spinal cord complications. An average of 2066 ml of blood was auto-transfused (range, 450-6100 ml). During the first 30 postoperative days, 17 patients (12.9 %) died. Eleven patients (8.3%) had spinal cord dysfunction, six patients (4.5%) had lower-extremity paraparesis, and five patients (3.8%) had paraplegia. Nine patients (6.8%) had renal failure necessitating hemodialysis. Other complications included bleeding in 15 cases (11.4%), respiratory failure in 12 cases (9.1%), wound-related sequelae in five cases (3.8%), distal embolism in five cases (3.8%), and bowel ischemia in two cases (1.5%). CONCLUSION: The single-clamp technique yielded an acceptable complication rate, and the mortality was comparable to that seen after the use of more complex methods. For satisfactory results, the cross-clamp time should not exceed 30 min.

Adult↗
PubMed1997

The de-airing clamp in cardiac surgery.

De-airing of the heart in open heart surgery is a necessary routine. Most of the air is evacuated from the heart before the aortic cross clamp is removed, but the de-airing may be continued even after declamping. One way to continue de-airing is to partially clamp the ascending aorta, proximally to the aortic cannula, in order to create a pocket for trapping residual air coming from the left ventricle. This paper describes a clamp specially designed and developed for this purpose. It has been used in our center since 1990 and our experience is reported. The clamp has been used on 250 patients and in 50% ultrasonography has been used to examine the heart being free from air bubbles within 20 min from releasing the aortic cross clamp.

Cardiac Surgical Procedures↗
PubMed1997

Single aortic cross-clamp technique in coronary surgery: a prospective randomized study.

OBJECTIVE: To test the hypothesis of an improved myocardial and cerebral protection by combining blood cardioplegia and the single aortic cross-clamp technique, 100 patients were enrolled in a prospectively randomized study and stratified for preoperative conditions. METHODS: In Group I, 55 patients underwent myocardial revascularization using crystalloid cardioplegia and the conventional partial occluding clamp technique to perform proximal anastomoses, whereas in Group II, 45 patients were operated on combining blood cardioplegia and the single aortic cross-clamp technique. Unstable angina, emergency procedures, reoperations and preoperative counterpulsation accounted for an higher risk score in group II patients (P < 0.03). Operations were performed by the same surgical team. Aortic cross-clamp time was significantly longer in group II patients (59 +/- 22 vs. 47 +/- 18 min.) (P < 0.001). Other intraoperative variables were not significant. RESULTS: A 70-year-old male in group I died on post-operative day 5 as a consequence of a major neurological event. Length of ventilatory dependency, post-operative bleeding, need for blood transfusions, ICU stay, and hospital stay were similar between the two groups (P = NS). Patients in group I showed a strict correlation between the duration of surgical ischemia and post-operative myocardial necrosis. Analysis of combined mortality and morbidity events (adverse events) between the two groups, led to a significant prevalence in group I patients (P < 0.03) in spite of an higher pre-operative risk score and longer ischemic times in group II patients. Neurological lesions remained confined to group I patients. CONCLUSIONS: The combined use of blood cardioplegia, delivered via the antegrade and retrograde routes, and the single-clamp technique to perform myocardial revascularization, might enhance myocardial and cerebral protection when compared to conventional methods. Larger groups of patients are needed to support this trend.

Adult↗