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An improved double vaseline gap voltage clamp to study electroporated skeletal muscle fibers.

An improved voltage clamp with a double vaseline gap chamber was designed to study electroporated skeletal muscle fibers. The new clamp eliminated spike overshock of membrane potential when applying step stimulation occurring in the traditional configuration. It allowed greater consistency in membrane potential distribution. After the intracellular resistances of the fiber segment at the vaseline gap area were compensated, it was possible to change membrane potential more quickly. Using this technique, strong electrical pulses used to mimic the situation of electrical shock can be delivered to the cell membrane by voltage clamp. Transmembrane currents of skeletal muscle cell were simultaneously measured during a high pulsed shock and resolved into different components. Distinct transient changes of the transmembrane current, involving the time courses of the formation of electroporation and their recovery time constants, can be recorded. Because of more even membrane potential distribution and faster response to pulsed membrane potential change, this technique is also suitable for membrane study under physiological conditions.

Animals↗

Single-channel electrophysiology: use of the patch clamp.

The patch clamp technique affords unparalleled resolution of the detailed properties of ion channel currents. Patch clamping is not difficult and has been used to record single-channel currents from many cell types. The number of artifacts associated with the method appears to be rather small. The main experimental difficulties arise from the need to process large amounts of data. With the development of inexpensive computers and mass storage devices, these problems should be alleviated. The study of membrane excitability is expanding rapidly owing to the introduction of high-resolution patch clamp techniques. The conceptual revolution that will inevitably follow is just beginning.

Animals↗

Crystal structure of the delta' subunit of the clamp-loader complex of E. coli DNA polymerase III.

The crystal structure of the delta' subunit of the clamp-loader complex of E. coli DNA polymerase III has been determined. Three consecutive domains in the structure are arranged in a C-shaped architecture. The N-terminal domain contains a nonfunctional nucleotide binding site. The catalytic component of the clamp-loader complex is the gamma subunit, which is homologous to delta'. A sequence-structure alignment suggests that nucleotides bind to gamma at an interdomain interface within the inner surface of the "C." The alignment is extended to other clamp-loader complexes and to the RuvB family of DNA helicases, and suggests that each of these is assembled from C-shaped components that can open and close the jaws of the "C" in response to ATP binding and hydrolysis.

Adenosine Triphosphate↗

Failure of cross clamping the thoracic aorta to control intra-abdominal bleeding.

The efficiency of cross clamping the thoracic aorta for cases of massive hemorrhage and impending cardiac arrest is questionable. We present two cases of major intra-abdominal aortic injury in which the bleeding could not be controlled by cross clamping the thoracic aorta. Multiple collateral pathways around the clamped aorta may have rendered this procedure almost ineffective. In cases of major intra-abdominal arterial disruption, emergency department laparotomy may be necessary.

Adult↗

Visceral lipid peroxidation occurs at reperfusion after supraceliac aortic cross-clamping.

PURPOSE: Recently, we have reported that lipid peroxidation specific to oxygen free radical-mediated injury increased immediately after reperfusion of human liver allografts. However, in the human liver transplantation setting it was impossible to disassociate the contributions to lipid peroxidation caused by the warm and cold ischemic phases from those caused by reperfusion. Therefore we now have studied lipid peroxidation at reperfusion after supraceliac aortic cross-clamping in patients with normal livers. METHODS: Ethane, a noninvasive biomaker of lipid peroxidation, was measured in exhaled breath of patients before and during cross-clamping of the thoracic aorta and at sequential time intervals after visceral reperfusion. RESULTS: Approximately a two-fold transient increase in the ethane level was observed at around 15 minutes after reperfusion in those patients whose aortas were cross-clamped for more than 18 minutes. CONCLUSIONS: These results indicate that free radical-mediated lipid peroxidation occurs at reperfusion of warm ischemic viscera in the clinical setting of aortic repair. This observation supports the hypothesis that substantial lipid peroxidation occurs when tissues are subjected to cold or warm ischemia followed by reperfusion.

Aged↗

Prevention of paraplegia during thoracic aortic cross-clamping: importance of patent internal mammary arteries.

PURPOSE: Unexplained anatomic and physiologic factors account for the unacceptably high rate of paraplegia/paresis after thoracoabdominal aortic reconstruction. We assessed the neurologic significance of patent internal mammary arteries (IMAs) in a novel rat model of aortic clamping in which the aortic origins of the intercostal arteries (ICAs) were occluded. METHODS: Twenty anesthetized, intubated, and halothane-ventilated adult male rats had catheters placed in the carotid and femoral arteries. ICAs arising from the aorta were divided at their origins through a left thoracotomy. IMAs were either divided (IMA-OUT, n = 10) or left intact (IMA-IN, n = 10). Proximal and distal descending aortic clamps were placed for 7 minutes. A neurologic deficit score (NDS) was assigned at 1, 4, 18, 24, and 48 hours by use of an established scoring system. RESULTS: The mean IMA-IN NDS was statistically better than the mean IMA-OUT NDS at 1, 4, 18, and 24 hours with p = 0.0005, 0.0014, 0.0098, and 0.0151, respectively. Moreover, the mortality rate in the IMA-OUT group was statistically greater than in the IMA-IN group (p = 0.0036). CONCLUSION: In this model, patent IMAs prevent paraplegia when the ICAs are occluded at their aortic origin and the aorta is clamped for 7 minutes.

Animals↗

Aortic stump closure with a titanium permanent clamp: a useful emergency method.

Successful aortic stump closure in a patient with Behçet's disease was accomplished with a permanent titanium clamp. In May 1990, a saccular infrarenal abdominal aortic aneurysm was detected in this patient, and prosthetic graft replacement was carried out. One year later, this graft was removed because of perigraft fluid collection; the aortic stump was sutured closed, and a right axillobifemoral bypass was done. In November 1994, the patient was admitted to the hospital because of an aortoenteric fistula. An emergency operation was performed, and the aortic stump was managed successfully with a permanent clamp. In patients with Behçet's disease, use of a permanent clamp may offer an alternative to traditional methods for closing blown-out aortic stumps.

Anastomosis, Surgical↗

[Hemodynamic effects of intranasal nifedipine during clamping of the abdominal aorta].

The haemodynamic changes due to cross-clamping of the abdominal aorta below the renal arteries were studied in ten patients. Anaesthesia was induced with thiopentone and maintained with fentanyl and vecuronium and inhalation of 60% nitrous oxide in oxygen. At the fifth minute, clamping increased mean arterial pressure (Pa) by 11%, systemic vascular resistance (Rsa) by 26% and decreased cardiac output (CO) by 20%. Nifedipine was administered intranasally at this time. Heart rate remained unchanged; mean pulmonary arterial and mean pulmonary wedge pressures were slightly decreased. Pa and Rsa fell to significantly lower levels between the fifth and fifteenth minutes (24 and 43% respectively). Although CO increased by 28%, this was not significant. The administration of intranasal nifedipine during anaesthesia was well tolerated. This study demonstrated that intranasal nifedipine prevented adverse haemodynamic effects of cross-clamping of the aorta below the renal arteries.

Administration, Intranasal↗

Comparison of cerclage techniques using a hose clamp versus monofilament cerclage wire or cable.

To assess the compressive force generated by different techniques of cerclage wiring, the compression produced by 6 different cerclage techniques, including monofilament wires, Dall-Miles cables (Howmedica, Rutherford, NJ), and hose clamps, was measured. Tests were performed on both a polysulfone cylinder model and a cadaver bone model. The hose clamp generated significantly higher compression force than any other cerclage device tested. Doubled wires produced similar compressive force as the cables. As the hose clamp is the least expensive and produces the maximum compression, it may be advantageous in instances in which maximum compression is desirable as a temporary device, but because it is not implantable, it must be replaced by other techniques prior to wound closure. The Dall-Miles cable is expensive and its compressive force can be equaled at lower cost by a doubled wire. The use of a single strand of monofilament wire was the least effective.

Bone Wires↗

Cerebrospinal fluid production during proximal aortic cross-clamping.

During cross-clamping (XC) of the thoracic aorta, the cerebrospinal fluid pressure (CSFP) increases. The mechanism of the increased CSFP is unknown but increased cerebrospinal fluid (CSF) production has been suggested as one explanation. The aim of this study was to investigate whether the CSF production rate was influenced by proximal XC of the thoracic aorta in an experimental model. Using the ventriculocisternal perfusion method including [14C] inulin, the rate of CSF production was measured before, during and after XC of the thoracic aorta in seven pigs. The CSFP was measured via a catheter in the lateral ventricle and the thoracic aorta was cross-clamped just distal to the left subclavian artery for 30 minutes. Following cross-clamping the CSFP increased from 5 mmHg to 11 mmHg (p < 0.001) and remained elevated during XC. In contrast the CSF production rate was 0.09 ml/min prior to XC and was not significantly altered following XC. In conclusion this experimental study indicates that increased CSF production is not responsible for the increase in CSFP during XC.

Animals↗

Intricacies in ATP-dependent clamp loading: variations across replication systems.

DNA replication requires the coordinated effort of many proteins to create a highly processive biomachine able to replicate entire genomes in a single process. The clamp proteins confer on replisomes this property of processivity but in turn require clamp loaders for their functional assembly onto DNA. A more detailed view of the mechanisms for holoenzyme assembly in replication systems has been obtained from the advent of novel solution experiments and the appearance of low- and high-resolution structures for the clamp loaders.

Adenosine Triphosphate↗

Ischemic spinal cord injury induced by aortic cross-clamping: prevention by riluzole.

OBJECTIVE: Recent studies confirmed the deleterious role of glutamate in the pathophysiology of spinal cord ischemia induced by aortic cross-clamping. We investigated the effect of riluzole, an anti-glutamate drug, in a rat model of spinal cord ischemia. MATERIALS AND METHODS: Spinal cord ischemia was induced in normothermia for 14 min in Sprague-Dawley rats using direct aortic arch plus left subclavian artery cross-clamping through a limited thoracotomy. Experimental groups were as follows: sham-operation (n=15), control (n=15) receiving only vehicle, riluzole (n=15) receiving riluzole (4 mg/kg) before clamping and at the onset of reperfusion. Separate animals were used for monitoring physiologic parameters in the sham-operation (n=3), control (n=5), and riluzole (n=5) groups. Neurologic status was assessed at 6, 24 h, and then daily up to 96 h. Rats were randomly killed at 24, 48, or 96 h (n=5 for each time). Spinal cords were harvested for histopathology, immunohistochemistry for microtubule-associated protein 2 (MAP-2), TUNEL staining, and analysis of DNA fragmentation by agarose gel electrophoresis. RESULTS: All sham-operated rats had a normal neurologic outcome, whereas all control rats suffered severe and definitive paraplegia. Riluzole-treated rats had significantly better neurologic function compared to the control. Histopathology disclosed severe neuronal necrosis in the lumbar gray matter of control rats, whereas riluzole-treated rats suffered usually mild to moderate injury. Riluzole particularly prevented motor neurons injury. MAP-2 immunoreactivity was completely lost in control rats, whereas it was preserved either completely or partly in riluzole-treated rats. TUNEL staining revealed numerous apoptotic neurons scattered within the whole gray matter of control rats. Riluzole prevented or dramatically attenuated apoptotic neuronal death in treated rats. DNA extracted from lumbar spinal cords of sham-operated and riluzole-treated rats exhibited no laddering, whereas spinal cords from control rats showed DNA laddering with fragmentation into approximately 180 multiples of base pairs. CONCLUSIONS: Riluzole may protect the spinal cord in a setting of severe ischemia by preventing neuronal necrosis and apoptosis. This drug may therefore be considered for clinical use during 'high risk' surgical procedures on the thoracoabdominal aorta.

Animals↗

Predictors of cerebrovascular events in patients subjected to isolated coronary surgery. The importance of aortic cross-clamping.

OBJECTIVE: Stroke is a major complication after coronary surgery, occurring in 1-4% of the patients. In this study, we evaluate the incidence and pre- and intraoperative risk factors for the development of a cerebrovascular accident (CVA) and the impact of such an event on perioperative mortality and on hospital length of stay. METHODS: Data from 4567 patients submitted to isolated coronary artery bypass grafting (CABG) with hypothermic ventricular fibrillation between 1992 and 2001 were entered prospectively into a dedicated computerized database and analyzed retrospectively at this time. Univariate and multivariate analyses were performed where appropriate. RESULTS: The incidence of postoperative CVA was 2.5% (116 patients). Multivariable logistic regression identified the following variables to be independent predictors of a postoperative CVA: cerebrovascular disease (P<0.001; odds ratio (OR), 2.66), peripheral vascular disease (P<0.001; OR, 2.33), number of periods of aortic cross-clamping (P=0.019; OR, 1.31 per each period of aortic cross-clamping), LV dysfunction (P=0.012; OR, 1.82) and age (P=0.008; OR, 1.28 per each 10 years). Non-elective surgery showed a marginal significance (P=0.08; OR 1.83). The 30-day mortality for patients who experienced a CVA was 16.4% versus 0.6% for patients who did not (P<0.001). Postoperative CVA increased the length of hospital stay threefold to 20.3+/-28.3 days as compared with patients who did not have a postoperative CVA (7.6+/-4.2 days; P<0.001). CONCLUSIONS: Postoperative CVA dramatically increases the mortality and length of stay after CABG. Identification of predisposing factors permits preoperative risk stratification and may facilitate improved patient selection or optimization. Our study adds evidence to the superiority of the fibrillation technique over intermittent cross-clamping of the aorta, among non-cardioplegic techniques, in terms of neurological protection.

Age Factors↗

Cardiac troponin T release during coronary surgery using intermittent cross-clamp with fibrillation, on-pump and off-pump beating heart.

OBJECTIVE: Troponin T is a unique marker which might be particularly useful in assessing myocardial cell damage in patients undergoing cardiac surgery. The aim of the study was a comparison between intra-operative release of troponin T (TnT) during three different myocardial protection strategies. METHODS: Thirty-eight PTS undergoing myocardial revascularization were randomised into 3 groups in whom procedure was performed with intermittent cross-clamp (Group I; n = 13), beating-heart on pump without aortic cross-clamp (Group II; n = 12), beating-heart without use of extracorporeal circulation (Group III; n = 13). Serial venous blood samples were collected for TnT measurement prior surgery, 1, 4, 12, 24, 48, 72 h after the procedure. Haemodynamic measurements were made using a thermodilution PA catheter. RESULTS: The groups were similar with respect to age, sex, preoperative LV function, number of grafts, potential risk factors. There were no hospital deaths and no myocardial infarction (MI) in three groups, postoperative haemodynamic measurements showed no significant differences. TnT serum levels were significantly higher in group I when compared to groups II and III. TnT levels were significantly lower in group Ill when compared to group II following 48-h post-operation. CONCLUSIONS: Coronary bypass grafting without aortic cross-clamping and without CPB offers superior myocardial protection.

Aged↗

Control of proximal hypertension during aortic cross-clamping: its effect on cerebrospinal fluid dynamics and spinal cord perfusion pressure.

Postoperative paraplegia remains the most devastating complication of surgery of the descending and thoraco-abdominal aorta. Control of the proximal hypertension that follows cross-clamping of the thoracic aorta to repain aneurysms of the descending and thoraco-abdominal aorta is necessary to prevent left ventricular failure, myocardial infarction, and hemorrhagic cerebral events. Both pharmacological and mechanical modalities used to control central hypertension during aortic occlusion affect cerebrospinal fluid dynamics and spinal cord perfusion pressure. Sodium nitroprusside (doses >5 microg/kg/min), the most widely used pharmacological agent, decreases spinal cord perfusion pressure because it increases cerebrospinal fluid pressure and decreases blood pressure distal to the aortic cross-clamp. This effect cannot be prevented by drainage of cerebrospinal fluid. Nitroglycerin also decreases spinal cord perfusion pressure, but its effects on cerebrospinal fluid dynamics can be countered by drainage of cerebrospinal fluid. Active distal perfusion with left atrial-femoral artery bypass can provide adequate perfusion of the circulation distal to the aortic cross-clamp while simultaneously reducing cerebrospinal fluid pressure. This approach can maintain mesenteric and spinal cord blood flow, therefore preventing the multiple organ dysfunction syndrome caused by release of cytokines from the splanchnic district and decreasing the incidence of postoperative paraplegia from spinal cord ischemia. In cases of limited retroperfusion, partial exsanguination and cerebrospinal fluid drainage can be used in conjunction with left atrial-femoral artery bypass to prevent rises in cerebrospinal fluid pressure and maintain spinal cord blood flow above the threshold necessary to prevent neurological injury. The use of oxygenated perfluorocarbons in the subarachnoid space to provide passive oxygenation of the spinal cord during aortic occlusion remains experimental and requires further investigation.

Antihypertensive Agents↗

Carotid endarterectomy monitoring: patterns of spectral EEG changes due to carotid artery clamping.

OBJECTIVE: The purpose of the study was to make an objective and quantitative analysis of the EEG changes caused by carotid artery clamping during carotid endarterectomy (CEA) monitoring. METHODS: Factor analysis was used to study the intraoperative spectral EEG changes in 94 patients during clamping of the carotid artery. In addition, the relation between the extracted factors and the changes in blood pressure and blood flow velocity in the middle cerebral artery during clamping was studied. RESULTS: Two factors were extracted with factor analysis. The first factor represented a change in power in the alpha and beta frequency ranges in combination with a less pronounced opposite change in power in the delta frequency range. The second factor represented a change in power restricted to the delta and theta frequencies. With the first factor, two types of spectral EEG changes could be distinguished: changes indicative of cerebral ischemia (decrease in fast activity and increase in slow activity) and the opposite changes suggesting cerebral activation (arousal). With the two factors combined, the changes indicative of minor ischemia (decrease in fast activity only) could also be distinguished. CONCLUSION: Further study is required to test whether patients showing the EEG changes indicative of activation or minor ischemia actually require shunting.

Aged↗

Stabilization of high-resistance seals in patch-clamp recordings by laminar flow.

The formation of a high-resistance electrical seal between a cell membrane and a glass micropipet tip is essential in patch-clamp experiments. We have studied the electrical properties and the mechanical stability of the seal using a microfluidic chip generating laminar flow in open volumes. We show that, by using fluid flow (1-10 mm/s) acting along the symmetry axis of the cell-pipet, seals of a higher mechanical stability with increased resistances can be achieved, allowing up to 100% longer recording times and over 40% decreased noise levels (Irms). These improved properties are beneficial for high-sensitivity patch-clamp recordings, in particular, in longtime studies of ion channel receptor systems that are relevant in biosensor applications of the technique. Furthermore, these observations support the combination of patch-clamp with microfluidic devices, for example, for rapid solution exchange around a single cell sensor for high-throughput electrophysiology and for highly resolved kinetic studies.

Journal Article↗