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PubMed1986

Patch clamp technique and biophysical study of membrane channels.

The present work describes the patch clamp technique, which first allowed the recording of single channel currents in biological membranes. In particular, it describes procedures for preparation and applications of the four different patch clamp configurations. Briefly, the cell-attached configuration is widely used for investigating channel modulation by transmitters acting via second messengers. The cell-free configurations (inside-out and outside-out), complementary to one another with respect to the orientation of the membrane surface, are particularly indicated for the study of the biophysics (kinetics, conductivity, selectivity, mechanism of permeation and block) of ionic channels. Finally, the whole-cell configuration which, because of the remarkable feature that it allows voltage clamp of very small cells, has given access to a number of physiologically important preparations never studied before.

Animals↗

PubMed1985

Patch and whole-cell voltage clamp of single mammalian visceral and vascular smooth muscle cells.

Dispersal of the constituent cells of mammalian visceral and vascular smooth muscles has permitted recordings both of membrane currents under whole-cell voltage clamp, and of currents through single ionic channels using the patch-clamp technique. A rectangular depolarizing step applied to a single cell under voltage clamp yielded a net inward current followed by a net outward current in normal physiological solution. In isolated, 'inside-out' patches of cell membrane a calcium- and potential-sensitive K channel (100 pS conductance) and a calcium-insensitive, potential-sensitive K+ channel (50 pS conductance) with slow kinetics have so far been identified and characterized.

Animals↗

PubMed1994

The efficacy of non-clamping selective cerebral perfusion in distal aortic arch aneurysm repair: report of a case.

Successful repair of a distal aortic arch aneurysm without aortic cross-clamping was carried out in a 74-year-old man, using a combination of special separate cerebral perfusion and retrograde coronary perfusion, termed non-clamping selective cerebral perfusion. We believe that satisfactory results following aortic arch surgery in elderly patients can only be achieved through the prevention of emboli derived from an aortic cross-clamping site, and shortened ischemic time of the vital organs.

Aged↗

PubMed1998

Timing of lymphocyte transfusion and portal clamping for the development of lethal graft-versus-host disease in the rat.

The effects of surgical intervention on the incidence and augmentation of graft-versus-host disease (GVHD) were studied in the rat. To elicit GVHD, splenocytes from parental LEW rats at a dose of 4 x 10(8) or 1.5 x 10(8) cells/350g body weight were injected via the tail vein into (LEW x BN)F1 rats. The injection of 4 x 10(8) of LEW rat splenocytes induced lethal GVHD in all nontreated F1 rats, while 1.5 x 10(8) of LEW splenocytes did not induce any signs of GVHD. However, when the F1 rats had received the same dose of parental LEW lymphocytes in combination with portal clamping, 14 recipients out of the 15 rats (93%) suffered GVHD and 10 rats (67%) died from GVHD. Interestingly, portal clamping 7 days after the injection enhanced the incidence of GVHD, whereas no GVHD was observed in the intervention group either 3 or 7 days prior to the cell transfusion. When 1.5 x 10(8) of allogeneic lymphocytes were injected intravenously together with 0.1-1.0 mg/kg of endotoxin instead of portal clamping, the injection led the early death by GVHD, while the injection of methylpredonisolone did not enhance GVHD. These results thus indicate that either simultaneous or delayed surgical intervention has the potential to trigger a dormant state in transferred alloreactive lymphocytes.

Animals↗

PubMed2002

An evaluation of coronary artery bypass grafting without aortic cross-clamping due to severely atherosclerotic ascending aorta.

OBJECTIVES: We evaluated coronary artery bypass grafting (CABG) in which aortic cross-clamping is not done due to severe atherosclerosis of the ascending aorta. METHODS: Subjects were 51 patients undergoing CABG without aortic cross-clamping during cardiopulmonary bypass under moderately hypothermic ventricular fibrillation in the 12 years from June 1988 to October 1999 (Group N). In some cases, empty beating or moderate hypothermic circulatory arrest was used. We compared these 51 with 1104 subjects undergoing conventional CABG with aortic cross-clamping and cardioplegic cardiac arrest in the 9 years from June 1988 to December 1997 (Group A). RESULTS: In all 6 cases with neurologic deficits, moderately hypothermic circulatory arrest was used during proximal anastomosis of saphenous vein grafts. Postoperative computed tomography scan showed them to have suffered infarction due to embolization. Multivariate analysis identified proximal saphenous vein grafting under moderately hypothermic circulatory arrest as a predictor of neurologic deficit. Complete revascularization was significantly lower in Group N. Actual survival and freedom from cardiac death were significantly lower in Group N. CONCLUSION: Manipulation of the atherosclerotic ascending aorta under moderately hypothermic circulatory arrest or ventricular fibrillation generates the highest risk of perioperative neurologic deficit and should thus be avoided. In-situ arterial grafting should be conducted with utmost care.

Adolescent↗

PubMed2000

A randomised controlled trial of delayed cord clamping in very low birth weight preterm infants.

UNLABELLED: This study was carried out to assess the feasibility of late cord clamping of 45 s in preterm infants delivered mainly by caesarean section and the effects on postpartal adaptation and anaemia of prematurity. Prior to delivery, 40 infants of < 33 gestational weeks were randomised to either 20 s or 45 s of late cord clamping. After the first shoulder was delivered, oxytocin was given intravenously to the mother in order to enhance placento-fetal transfusion while the infant was held below the level of the placenta. The 20 infants in group 1 (20 s) had a mean birth weight of 1070 g and a mean gestational age of 29 + 4/7 weeks versus 1190 g and 30 weeks in group 2 (45 s). On day 42 of life there were ten infants without transfusions in group 2 versus three in group 1 (P < 0.05). Out of the 20 infants in group 1, 19 and 15/19 in group 2 were delivered by caesarean section. There were no significant differences in Apgar scores, temperature on admission, heart rate, blood pressure and requirements for artificial ventilation. CONCLUSION: Delayed cord clamping of 45 s is feasible and safe in preterm infants below 33 weeks of gestation. It is possible to perform the procedure at caesarean section deliveries and it should be performed whenever possible. It reduces the need for packed red cell transfusions during the first 6 weeks of life.

Anemia, Neonatal↗

PubMed1990

The incorporation of 5-fluorouracil into rat liver tumor and normal tissues after administration by the hepatic artery during temporary portal vein clamping.

A therapeutic dose of labelled 5-fluorouracil (5-FU) was infused via the hepatic artery during 30 min with or without a simultaneous temporary clamping of the portal vein in a model of secondary liver cancer in Wistar rats. After another 60 min, the incorporation of 5-FU into the acid soluble fraction, RNA and DNA was determined in tumor, liver, small intestine, kidney, and bone marrow. The liver and intestinal nucleotide profiles were examined with isotachophoresis. Temporary portal vein clamping caused the following changes, as compared with the control group with intraarterial infusion only: in the liver, the incorporation of 5-FU into the acid soluble fraction increased without a concomitant increase into the RNA or of the level of (F)UTP. Liver ATP decreased. In the tumor, the ratio of RNA to acid soluble fraction labelling decreased. There was a generally decreased ratio of tumor to peripheral normal tissue (small intestine and kidney) labelling. In conclusion, portal vein clamping in conjunction with hepatic arterial administration of 5-FU led to a decreased anabolism of 5-FU in the liver and tumor, and increased systemic drug exposure. It is not known how this interferes with the therapeutic effect.

Adenosine Triphosphate↗

PubMed2005

Postoperative evolution of inflammatory response in a model of suprarenal aortic cross-clamping with and without hemorrhagic shock. Systemic and local reactions.

Surgery of the abdominal aorta generates a systemic inflammatory response (SIR), a source of operative morbidity-mortality. In the present work we attempted to evaluate the evolution of SIR in an experimental model that simulates elective and urgent surgery on the abdominal aorta. Fifteen mini-pigs divided into three groups were used. The animals were subjected to suprarenal aortic/iliac clamping and bypass with a Dacron-collagen prosthetic graft. Groups were as follows: (1) sham (only aortic dissection); (2) clamping and bypass; (3) hemorrhage of 40%, pre-clamping, and bypass. Determinations included (1) tumor necrosis factor-alpha (TNF-alpha) interleukin (IL)-1beta, IL-6, IL-10, interferon-gamma; (2) myeloperoxidase (MPO), superoxide anion (SOA), superoxide dismutase (SOD), and malondialdehyde (MDA); (3) nitrites; (4) iNOS, (5) cell adhesion molecules (ICAM-1, VCAM-1) at 24 hours, 48 hours, and on day 7; and (6) NFkappaB at 48 hours. Our results point to an increase in all inflammatory variables, corroborated by their molecular regulators such as the expression of CAMs, iNOS, and NFkappaB. The alterations tended to normalize by day 7, after reperfusion. The results point to the great importance of SIR at all levels (molecular, nuclear, cellular, and systemic) in situations such as elective and urgent abdominal aorta surgery and the role that control of this response could represent for the future of vascular surgery.

Animals↗

PubMed2004

Adaptation of skull clamp for use in image-guided surgery of children in the first 2 years of life.

INTRODUCTION: We describe a simple but effective modification of the skull clamp, aimed at stabilising the head of very young children, while avoiding the risk of creating a depressed skull fracture, in order to enable the utilisation of image-guidance in such young patients. METHODS: We machined three small perspex discs 3 cm in diameter. On the outer surface of these pads we drilled reception holes for the pins to prevent slippage. To avoid direct contact with the skin, we interfaced a thick pad of soft felt. During intraoperative positioning, the weight of the head was supported by a suction bean-bag placed on the operating table. Hence, the clamp apparatus was employed only to secure the head position, and not to support the weight of the head, thus requiring less clamp force. We employed this modification in three children (aged 9, 13 and 15 months) who required image-guided surgery for brain tumours. OUTCOME: In all cases the head remained immobile throughout the operation, making possible the accurate use of image guidance. At the end of the operation, some transient skin redness was noticed in the contact areas, which settled in a few days.

Brain Neoplasms↗

PubMed1998

Titanium-nickel shape memory clamps in small bone surgery.

The therapeutic applications of titanium-nickel (TiNi) shape memory clamps with optimized structural characteristics for fixation of small bone fragments in 64 patients are presented. Our fixative makes compressive stabilization possible and enables a patient to start rehabilitation very soon after surgery. The supporting splint immobilization never exceeded 12 days. Three aspects were studied: bone union, wound healing problems and histology. Non-union occurred in 4 patients treated with only one fixative. Two clamps implanted in non-parallel planes seem to be advisable to exclude the need for longer immobilization. Neither toxic manifestation nor episodes of an allergic reaction occurred. No suppuration appeared when a heat stimulus was supplied by using a contact resistance heater. Histological evaluation of the tissue covering the implants in 22 patients did not reveal any adverse reaction. Our study suggests that by using TiNi clamps in an appropriate way, satisfactory outcomes could be achieved with respect to both biofunctionality and biocompatibility.

Alloys↗

PubMed2006

Cerebral ischemia during carotid artery cross-clamping: predictive value of phase-contrast magnetic resonance imaging.

The goal of this prospective study was to determine the utility of preoperative cerebral magnetic resonance imaging (MRI) in predicting cerebral ischemia during carotid artery cross-clamping for endarterectomy. Between January 2000 and December 2003, a total of 121 patients (95 men, 26 women) underwent three-dimensional phase-contrast MRI to assess collateral function prior to carotid endarterectomy. During regional anesthesia, patients were monitored to detect ischemic events and their timing in relation to cross-clamping and to determine mean intraoperative arterial pressure. These findings were then correlated with the collateral variations observed in the circle of Willis on preoperative MRI. Patients were classified into three groups according to neurological tolerance: normal tolerance (n = 106), immediate severe deficit (n = 9), and late deficit associated with arterial hypotension (n = 6). In the second group, a significant correlation was found between the absence of collateral circulation and neurological deficit (p < .0001). These results indicated that three-dimensional phase-contrast MRI is useful for predicting cerebral ischemia during carotid cross-clamping and selecting indications for shunting. Absence of visible collaterals of the circle of Willis on MRI is significantly predictive of early ischemia and an indication for systematic shunt placement.

Aged↗

PubMed1975

Cesarean section prevents placenta-to-infant transfusion despite delayed cord clamping.

We have adapted and calibrated an accurate method for measuring residual placement blood volume (RPBV). We used this method in 33 normal vaginal deliveries and 18 cesarean section deliveries in which the umbilical cord was clamped from 5 to 118 seconds after delivery. Infants born vaginally received 10 to 20 ml. per kilogram of body weight of placental transfusion when umbilical cord was not clamped until 30 seconds or more after delivery. Equivalent delay of umbilical cord clamping in cesarean section infants produced no placental transfusion.

Birth Weight↗

PubMed1989

A method for preparing freeze-clamped tissue samples for metabolite analyses.

A rapid and simple method for preparing freeze-clamped tissue samples for metabolite determinations is described. Freeze-clamped rat heart tissue samples weighing from 0.8 to 1.0 g were homogenized directly in an Ultra-Turrax homogenizer for 60 s in 3.5 ml of ice-cold 0.6 M HClO4 without pulverizing them in liquid nitrogen. After centrifugation, the pellet was rehomogenized in the Ultra-Turrax homogenizer for 30 s in 1.5 ml of HClO4. Following a further centrifugation the extracts were combined and the pH was adjusted to 7.0 by adding 5 M K2CO3. The neutralized supernatant was used for the desired assays. The analyses of the tissue extracts obtained from isolated perfused rat hearts by the present method give similar results for different kinds of metabolites than those processed according to the previous classical method. Moreover, the values of the various parameters determined from the tissue extracts prepared according to the method described here are similar to the data reported in literature. The method can be readily applied to any other freeze-clamped tissue. The greatest improvement obtained is that the homogenization procedure can be accomplished easily and conveniently in about one-tenth of the time required for the earlier classical method without the time-consuming and unpleasant tissue grinding in liquid nitrogen.

Adenosine Triphosphate↗

PubMed1994

Single-clamp technique: an important adjunct to myocardial and cerebral protection in coronary operations.

To determine the myocardial and cerebral protective properties of the single cross-clamp (group I; n = 160) versus the partial occluding clamp (group II; n = 150) technique for construction of the proximal anastomoses, a retrospective analysis of 310 patients operated on by the same surgeon was performed. Group I patients were older (median age, 70 versus 64 years; p < or = 0.0001), with 83 (52%), versus 41 (27%) in group II, 70 years and older (p < or = 0.0001). More group I patients were in New York Heart Association functional class IV (42 [26%] versus 22 [15%]; p = 0.008); more required preoperative balloon counterpulsation (35 [22%] versus 16 [11%]; p = 0.006); and more required emergent operation (20 [13%] versus 3 [2%]; p < or = 0.0001). Antegrade crystalloid cardioplegia was used in both groups. The median cross-clamp time was 58 minutes for group I versus 44 minutes for group II (p < or = 0.0001). However, there was no significant difference between the two groups in terms of the number of bypass grafts, the use of the mammary artery, or the bypass time. The operative mortality was 2.5% (n = 4) for group I versus 5.3% (n = 8) for group II (p = 0.16), and the perioperative myocardial infarction/low cardiac output state was seen in 6 patients (3.8%) in group I versus 18 patients (12%) in group II (p = 0.006). The median creatine kinase MB release was 13 U/L for group I versus 19 U/L for group II (p = 0.0029).(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1996

Effect of nitroglycerin on spinal cord ischemia after thoracic aortic cross-clamping.

BACKGROUND: Thoracic aortic cross-clamping with the use of sodium nitroprusside (SNP) has been shown to cause a decrease in spinal cord perfusion pressure and an increased incidence of paraplegia. Nitroglycerin is frequently used in this setting. This study investigated the effects of nitroglycerin and SNP on spinal cord ischemia. METHODS: Three-groups of 8 mongrel dogs underwent thoracic aortic cross-clamping for 45 minutes. Proximal pressure was maintained between 95 and 100 mm Hg with SNP, nitroglycerin, or phlebotomy. All animals were neurologically evaluated 24 hours later by a blinded observer, and the findings were confirmed by histopathologic study. Statistical analysis (p value of less than 0.05) of measured hemodynamic data was by analysis of variance and of Tarlov scores, the Mann-Whitney U test. RESULTS: Distal aortic pressures (p < 0.001), Tarlov scores, and spinal cord perfusion pressures (p < 0.01 and p < 0.05 for SNP group and nitroglycerin group, respectively) were significantly higher in the phlebotomy group compared with the SNP and NTG groups. Cerebrospinal fluid pressures were significantly lower in the phlebotomy group compared with the SNP group (p < 0.001). CONCLUSIONS: The use of either NTG or SNP was associated with a high incidence of paraplegia. Nitroglycerin appears to be no safer than SNP when used during thoracic aortic cross-clamping.

Animals↗

PubMed1993

Effect of sodium nitroprusside on paraplegia during cross-clamping of the thoracic aorta.

Sodium nitroprusside (SNP) is usually used to control excessive proximal pressure after aortic cross-clamping. To assess the effect of SNP on circulatory dynamics, somatosensory evoked potentials, and neurologic outcome, 10 adult mongrel dogs that underwent 45 minutes of cross-clamping of the thoracic aorta were randomly assigned to receive either 50 mg/kg of SNP or no treatment for excessive proximal hypertension. There was a statistically significant difference noted between the SNP-treated animals and the control animals in terms of the proximal mean arterial pressures (112 +/- 13 versus 142.2 +/- 15 mm Hg, respectively; p < 0.05) and the mean distal arterial pressures (15 +/- 3 mm Hg versus 23 +/- 1 mm Hg; p = 0.04). However, the electrical activity of the spinal cord, as indicated by the somatosensory evoked potentials, returned significantly faster in the nontreated group than in the SNP-treated group (15 +/- 9 versus 44 +/- 13 minutes; p < 0.05). Control animals exhibited a significantly better neurologic outcome and no paraplegia 24 hours postoperatively. We conclude that the use of SNP to treat excessive proximal hypertension may be detrimental to the spinal cord during cross-clamping of the thoracic aorta, resulting in a decline in the ischemic tolerance.

Animals↗

PubMed1976

Voltage-clamp experiments on oxidized cholesterol membranes modified with excitability-inducing material and comparison with a model.

The conductance of oxidized cholesterol membranes modified with excitability-inducing material was observed in membranes containing either single conductance channels or 100-1000 channels. Membranes containing single channels have several conductance states for each voltage polarity, and the current through membranes containing manychannels decays with at least two, and probably three, time constants following a step change in voltage (voltage-clamp). The time constants differ by about an order of magnitude. The multi-state behavior seems more pronounced in membranes made from highly oxidized cholesterol. Although a given conductance state could occur at either positive or negative voltages, each state was much more frequent at one polarity or the other. The most frequently observed single-channel conductance states in 0.1 M NaCl are about 0.3, 0.1, 0.03, and 0.0 n-1 for negative voltages and 0.25, 0.05, 0.03, and 0.0 n-1 for positive voltages. The current following a voltage clamp decays to a quasi-steady state within 1 min for positive voltages and 1-15 min for negative voltages. When the holding voltage is --20 mV, the decay constants and quasi-steady state conductances as functions of clamping voltage are reasonably well described by either a three-state model of the conductance or a two-state model applied independently at negative and positive voltages. However, for high voltages, the quasi-steady state does not appear to approach a state in which all the channels are in a low conductance state.

Cholesterol↗

PubMed1976

The effects of pentylenetetrazol on molluscan neurons. II. Voltage clamp studies.

The effects of pentylenetetrazol (PTZ) upon the steady and transient outward ionic currents during PTZ-induced prolonged depolarizations were investigated using voltage clamp techniques. PTZ causes a 5-35% reduction in gL and a 40-60% reduction in steady-state gK. There is also a marked reduction in the activation of gA of Connor and Stevens6 at all clamp potentials; a shortening of the time constant for the inactivation of gA; and a 10-15 mV shift in the depolarizing direction of the curve relating the steady-state inactivation of gA to membrane potential. The equilibrium potentials for both gA and gK are depolarized by 20 mV in PTZ solution. Equation and voltage clamp data for normal repetitive firing were integrated with the normal and PTZ-alered data. Solution to these equations demonstrated: (1) normal repetitive firing in response to a constant current stimulus; and (2) PTZ-altered repetitive firing that was in the direction of, and for the most part, similar to the observed behavior.

Animals↗