Cold Isotonic Saline Research Articles

Preservation of the Inferior Mesenteric Artery With a Surgeon-customised Fenestration

Abdominal aortic aneurysms (AAA) are most frequently treated by endovascular aneurysm repair (EVAR). A recognised drawback of EVAR is loss of the inferior mesenteric artery (IMA), which when associated with the additional occlusion of both hypogastric arteries, puts the patient at risk of ischaemic colitis or pelvic ischaemia. A surgeon-customised fenestration technique used in a 70 year old man with an AAA, occluded hypogastric arteries, and a large IMA is described. The diameter of the IMA, distance to the lower renal artery, and clock face positions were assessed with TeraRecon software (TeraRecon Foster City, CA, USA). The main body of an Endurant stent graft (Medtronic, Dublin, Ireland) was partially unsheathed. The location and shape of the fenestration was drawn on the graft between two rows of stent. A 6 mm fenestration was created by using a 1205 °C cautery loop tip (Accu-Temp; Beaver-Visitec International, Waltham, MA, USA). To prevent the burning of the polyester, the graft was continuously irrigated with cold isotonic saline. A segment of a 25 mm Amplatz goose neck snare (Medtronic) was sutured around the fenestration with a polytetrafluoroethylene monofilament suture (CV-5 Gore-Tex; WL Gore & Associates, Flagstaff, AZ, USA) to serve as a radio-opaque marker (Fig. 1A). The graft was then re-sheathed by using a silicone rubber band tightly wrapped around the graft and loosened gradually when the sheath came over (Fig. 1B). Once the stent graft was deployed, the fenestration and the IMA were catheterised with a SIM1 catheter and a 7 × 22 mm covered stent (Advanta V12; Maquet, Sunderland, UK) (Fig. 2A and B) was subsequently deployed and flared with a 9 × 20 mm angioplasty balloon.Figure 2(A) Catheterisation of the inferior mesenteric artery by surgeon-customised fenestration. (B) Post-operative three dimensional reconstruction showing patency of the fenestration and occlusion of both hypogastric arteries.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Surgeon-customised fenestration is a straightforward technique that provides an option for maintaining IMA patency in patients at risk of ischaemic colitis and pelvic ischaemia following infrarenal EVAR.

Towards quantitative metabolomics of mammalian cells: Development of a metabolite extraction protocol

Metabolomics aims to quantify all metabolites within an organism, thereby providing valuable insight into the metabolism of cells. To study intracellular metabolites, they are first extracted from the cells. The ideal extraction procedure should immediately quench metabolism and quantitatively extract all metabolites, a significant challenge given the rapid turnover and physicochemical diversity of intracellular metabolites. We have evaluated several quenching and extraction solutions for their suitability for mammalian cells grown in suspension. Quenching with 60% methanol (buffered or unbuffered) resulted in leakage of intracellular metabolites from the cells. In contrast, quenching with cold isotonic saline (0.9% [w/v] NaCl, 0.5 °C) did not damage cells and effectively halted conversion of ATP to ADP and AMP, indicative of metabolic arrest. Of the 12 different extraction methods tested, cold extraction in 50% aqueous acetonitrile was superior to other methods. The recovery of a mixture of standards was excellent, and the concentration of extracted intracellular metabolites was higher than for the other methods tested. The final protocol is easy to implement and can be used to study the intracellular metabolomes of mammalian cells.

Haemodynamic consequences of mild therapeutic hypothermia after cardiac arrest

Mild therapeutic hypothermia (MTH) is used after out-of-hospital cardiac arrest (OHCA) to minimize cerebral damage. Induced hypothermia may further interfere with cardiac function and influence haemodynamics after OHCA. This was a prospective study of haemodynamic variables in 50 consecutive patients with OHCA treated with MTH. Patients were cooled to a core body temperature of 32.5 degrees C for 24 h. Induction and maintenance of cooling was accomplished via infusion of 2 l of cold isotonic saline (4 degrees C) and a cooling blanket. Rewarming was performed to 36 degrees C at a rate of 0.3 degrees C per hour. Haemodynamic data were analysed and compared in individual patients during different temperature phases. Heart rate dropped from a mean of 85 to 60 beats per min (P=0.001) during hypothermia. Mean arterial pressure dropped from 79 to 72 mmHg, despite a rise in vasopressors and inotropes. Lactate levels were elevated throughout the induction (mean+/-SD) and maintenance phase (mean+/-SD); however, this did not correlate with a decrease in SVO2. Pulmonary artery pressures decreased during induction of hypothermia despite rapid infusion. MTH after OHCA lowered the heart rate. Despite induction of hypothermia with cold fluids, filling pressures decreased. Lower mean arterial pressure and cardiac output were observed during MTH, without deleterious effect on ScVO2. Lactate levels were elevated during MTH; however, levels did not correlate with outcome. Although the need for vasopressors and inotropes increases, this hypothermia-induced metabolic beta-blocker-like effect seems to have no negative effect on oxygen consumption and only temporarily affects anaerobic metabolism. No association of haemodynamic changes during MTH with outcome was found.

EVIDENCE FOR ASCORBIC ACID TRANSPORT SYSTEM IN RAT BRAIN CAPILLARIES

Although ascorbic acid (AA) crosses the choroid plexus and may enter the brain at an appreciable rate, it is not clearly established that there exist transport system(s) carrying this vitamin from blood into the brain cells across the brain capillaries. Thus the rate of its uptake by choroid plexus and cerebral capillaries were evaluated in vitro in this study. Choroid plexus and brain capillaries were isolated from two-month-old male Sprague-Dawley rats. Time course of AA incorporation in micro vessels and choroid plexus was studied up to 30 min. After stopping the incorporation with the excess of cold isotonic saline, micro vessels were filtered and sonicated. The intracellular incorporated AA radioactivity was measured by liquid scintillation counting. AA uptake by micro vessel was tested for Na+-dependence and saturability. The time course studies showed linear increase in total uptake and accumulation of AA by choroid plexus and endothelial cells up to 30 min. Treatment with oubain or replacement with sodium chloride showed that uptake is an Na+- independent process. Transport of AA to cerebrospinal fluid and brain was also shown to be readily saturated by increasing the level of cold AA. These results document that the brain capillary endothelial cells are able to transport and accumulate AA, and may have a critical role in the homeostasis and regulation of cerebral ascorbic acid concentration.

Thermosensitivity of muscle: high-intensity thermal stimulation of muscle tissue induces muscle pain in humans.

Small-calibre afferent units responding to thermal stimuli have previously been reported to exist in muscle. The question as to whether these receptors in humans mediate subjective thermal sensations from muscle remains unresolved. The aims of the present study were to determine in humans whether intramuscular injection of warm and cold isotonic saline elicits temperature sensations, muscle pain or any other sensations. In 15 subjects, no thermal sensations assessed on a temperature visual analogue scale (VAS) could be detected with intramuscular injections of isotonic saline (1.5 ml) into the anterior tibial muscle at temperatures ranging from 8 to 48 degrees C. The same subjects recorded strongly increasing scores on a temperature VAS when thermal stimuli in the same intensity range were applied to the skin overlying the muscle by a contact thermode. However, I.M. isotonic saline of 48 degrees C induced muscle pain with peak scores of 3.2 +/- 0.8 cm on a VAS scale ranging from 0 to 10 cm. Using the the McGill pain questionnaire a subgroup, of subjects qualitatively described the pain using the 'thermal hot' and 'dullness' word groups. Temperature measurements within the muscle during the stimulating injections showed that the time course of the pain sensation elicited by saline at 48 degrees C paralleled that of the intramuscular temperature and far outlasted the injection time. The present data show that high-intensity thermal stimulation of muscle is associated with muscle pain. High-threshold warm-sensitive receptors may mediate the pain following activation by temperatures of 48 degrees C or more. Taken together, the data indicate that thermosensation from a given volume of muscle is less potent than nociception.

A differential bronchomotor response to cooling and drying the upper airway

The present study aimed to compare the reflex bronchoconstrictor effects of upper airway applications of dry airstreams with equivalent cooling produced by cold isotonic and hypertonic saline solutions in anaesthetized rabbits. A method of on-line, breath by breath analysis of lung mechanics was used. The changes in total lung resistance (R l) and dynamic lung compliance (Cdyn) produced by dry airstreams and by cold hypertonic saline were greater than those induced by cold isotonic saline. Warm isotonic saline and supersaturated airstreams were ineffective. These results implicate osmotic disturbances in the response to dry airstreams. The response to dry airflow was abolished by section of the superior laryngeal nerves, by vagotomy and by atropine, suggesting a central cholinergic reflex mechanism is involved. Phosphoramidon had no effect, indicating that tachykinins are unlikely to play a role in this particular reflex.

Evaluation of a new continuous thermodilution cardiac output monitor in critically ill patients

To evaluate the accuracy of a new continuous cardiac output monitor (one based on the thermodilution principle) in critically ill patients. Criterion standard study. Multidisciplinary intensive care unit in a university hospital. Fourteen critically ill patients, with different diseases, requiring pulmonary artery catheterization. In two patients with a left ventricular assist system, a defined, sudden 1 L/min change in cardiac output was carried through to evaluate the in vivo response time of the continuous cardiac output monitoring system. In the remaining 12 patients, cardiac output was altered by varying the dose of catecholamines, by volume loading, or by varying the level of sedation. In four patients, a rapid infusion of cold saline was given through a central venous catheter to test the performance of the system under these conditions. Cardiac output was monitored continuously. A total of 163 (13 to 18 per patient) bolus determinations of cardiac output were performed, using the conventional thermodilution technique and simultaneously using the indocyanine green dye dilution technique. The range of cardiac output was 3.8 to 15.6 L/min. The results of the continuous thermodilution method were compared with the results of the bolus thermodilution and the dye dilution methods, respectively. The mean difference (bias) +/- SD of differences (precision) was 0.35 +/- 1.01 L/min for continuous vs. bolus thermodilution and 0.34 +/- 1.01 L/min for continuous thermodilution vs. indocyanine green dye dilution. Linear regression (correlation) analyses were y = 0.95x + 0.76 (r2 = .91) for continuous and bolus thermodilution and y = 0.93x + 0.87 (r2 = .91) for continuous thermodilution and dye dilution. The 75% in vivo response time was 10.5 mins. The infusion of cold isotonic saline led to erroneous continuous cardiac output values. When the conventional bolus thermodilution and dye dilution techniques were compared, mean difference was -0.01 +/- 0.54 L/min and the results of linear regression analyses were y = 0.97x + 0.22 (r2 = .97). Continuous cardiac output measurement using the thermodilution technique is reasonably accurate and is reliable and applicable in routine clinical practice, and therefore may add to patient safety. However, the response time is too slow for the immediate detection of acute changes in cardiac output. Some clinical conditions such as the rapid infusion of cold solutions can interfere with the continuous cardiac output measurement. Conventional bolus thermodilution and indocyanine green dye dilution methods showed good agreement and can be used interchangeably.

Quantitation of mucous glycoprotein removed from the respiratory tract of the rat by pulmonary lavage

Mucousglycoprotein (MGP) hypersecretion is a cause of chronic pulmonary obstructive disease and is also a common response of the pulmonary airways to toxicant-induced injury. To examine the feasibility of employing pulmonary lavage to assess the airway MGP content of the rat, the MGP removed from the lungs of male Sprague-Dawley rats by three successive lavages with cold isotonic saline were solubilized with urea and mercaptoethanol, and were purified by ultrafiltration and Sepharose CL-6B gel chromatography. MGP, which elute with the void volume of this gel, were quantitated by their carbohydrate content. Initial studies revealed that succeedings lavages removed succeedingly less MGP, suggesting that some pool of MGP was being washed out by this procedure. In a second study, isoproterenol was administered by a regimen known to produce MGP hypersecretion in the rat as assessed by histologic criteria (100 mg/kg/day, sc, for 6 days). Lavage MGP content of isoproterenol-treated rats averaged 450 μg compared to 210 μg in vehicle-treated controls ( p = 0.01). In summary, it is possible to purify and quantitate the MGP removed from the airways of the rat by pulmonary lavage and the amount of lavagable MGP is approximately doubled by isoproterenol, an agent known to induce the morphologic signs of mucous hypersecretion.

A radioreceptor assay in which iodocyanopindolol is used to determine propranolol and its active metabolites in unextracted serum or plasma.

This rapid, sensitive, simple radioreceptor assay (RRA) for l-propranolol and its active metabolites in unextracted samples requires 5 microL of sample, a beta-adrenergic antagonist, 125-l-labeled (-)cyanopindolol (125ICYP), and turkey erythrocyte membrane receptors (Kd = 40 pmol/L). Equal volumes (100 microL) of diluted sample and 125ICYP are incubated with 500 microL of erythrocyte membranes for 30 min. Cold isotonic saline (2.5 mL) is added, and the mixture is centrifuged. The concentration of drug that inhibited receptor binding by 50% (IC50) was l-propranolol, 1.5 micrograms/L; d-propranolol, 243 micrograms/L; and 4-hydroxypropranolol, 8.8 micrograms/L. Analytical recovery of propranolol added to serum was 93 to 120%; results for 138 clinical samples tested by this method and by liquid chromatography correlated well (r = 0.96). Results correlated strongly (r = 0.98) for 23 clinical samples tested by RRA and analyzed for both propranolol and 4-hydroxypropranolol by liquid chromatography. The sensitivity of this RRA was 0.3 micrograms/L, and both intra- and interassay CVs were less than 12%. Modifications of this method could permit testing of other nonselective beta-blockers.

Effect of diverting bile and pancreatic secretions into the ileum on small bowel mucosa in rats fed a liquid formula diet.

The long-term effects of diverting bile and pancreatic secretions directly into the ileum on small bowel mucosa was determined in rats fed a hydrolyzed liquid formula diet. Male Sprague-Dawley rats were divided into four experimental groups: duodenal papilla transplant-sham operation, transplantation of the duodenal papilla into the ileum, bile duct sham operation, and transplantation of the bile duct into the ileum. After 28 days, animals were killed, the same bowel removed, rinsed with cold isotonic saline, and divided into six segments (two jejunal segments and four ileal segments). The mucosa of each segment was weighed and assayed for DNA and protein concentration, and specific activity of sucrase and maltase. Bile and particularly pancreatic secretions diverted into the ileum stimulated local mucosal growth compared with their respective controls. The absence of pancreatic secretions from the jejunum also was associated with an increase in jejunal mucosal mass. Diverting pancreatic secretions into the ileum decreased ileal sucrase and maltase specific activity while the absence of both bile and pancreatic secretions from the jejunum increased jejunal sucrase specific activity. The results suggest that bile and pancreatic secretions entering the ileum are important factors in stimulating ileal mucosal hyperplasia while the absence of these secretions from the proximal intestine is associated with greater jejunal mucosal growth. The mechanisms regulating jejunal mucosal growth appear to be different than those influencing the ileum.

A rapid method for the isolation of glomeruli from the human kidney.

Uniformly decapsulated glomeruli were obtained from human kidney in less than 20 minutes. The kidney was perfused with cold isotonic saline solution. After removal of the medulla the cortex was cut into small fragments and forced through an 80-mesh stainless steel sieve. The cortical mash on the bottom surface of the sieve was then collected and suspended in 50 ml. cold saline solution. The cortical suspension was poured onto a 120-mesh wire cloth resting 10 cm. above a 200-mesh sieve. Each sieve was washed with 200 ml. saline solution. The material on top of the 200-mesh sieve, containing a pure preparation of glomeruli, was collected and centrifuged at 1,060 × g. for 3 min. The wet glomerular pellet was then buttered through a clean 200-mesh sieve and washed with 10 ml. saline solution. The emerging suspension contained uniformly decapsulated glomeruli with minimal glomerular disintegration. The suspension was centrifuged at 1,060 × g. for 2 min., the supernatant decanted and the residue lyophilized. The glomerular pellet, obtained from one human kidney, weighed 10 ± 3.0 mg.

HISTOCHEMICAL DEMONSTRATION OF PHOSPHOLIPASE B (LYSOLECITHINASE) ACTIVITY IN RAT TISSUES

A method has been developed for the histochemical demonstration of phospholipase B (lysolecithinase) of rat tissues. The enzyme attacks lysolecithin with liberation of 1 mole of glycerylphosphorylcholine and 1 mole of fatty acid. The recommended procedure involves use of 6-10 micro frozen sections, fixed in cold calcium-formol and incubated at 37 degrees C in Tris buffered medium at pH 6.6 containing 2.2 X 10(-3) M lysolecithin and 1% cobalt acetate. The fatty acid liberated by enzymatic hydrolysis is trapped as a cobalt precipitate and is then converted to a black-brown precipitate by treatment with dilute ammonium sulfide in cold isotonic saline. Equivalent amounts of fatty acid and glycerylphosphorylcholine are recovered by extraction and analysis of the incubated sections and of the incubation medium, thus proving that lysolecithin hydrolysis occurs under the proposed reaction conditions. Staining is reduced by treating the sections with copper ions, mercury compounds, alcohols, acetone and by heating at 60 degrees C prior to incubation with substrate. Lowering of the pH of the incubation medium has similar effect. These findings are interpreted as evidence of the enzymatic nature of the reaction. Cells exhibiting a positive staining are found in the lamina propria of the intestinal villi and crypts, in the red pulp of the spleen and in the interstitial tissue of lung, liver and thymus. Similar elements are present in bone marrow smears and in leukocyte preparations obtained by peritoneal lavage. The morphologic and staining characteristics of these cells correspond to those of the eosinophilic leukocytes. Physical and chemical agents (x-irradiation, corticosteroids) which sharply decrease the number of eosinophils also reduce the number of cells shown histochemically to hydrolyze lysolecithin. A correspondent diminution of phospholipase B activity of homogenates of the same tissues can be shown in vitro. Differences in tissue distribution and chemical properties distinguish the phospholipase B from less specific esterases and lipases.