Leo J. Menz

Cold Blood as the Vehicle for Potassium Cardioplegia

Cold blood with potassium, 34 mEq/L, was compared with cold blood and with a cardioplegic solution. Three groups of 6 dogs had 2 hours of aortic cross-clamp while on total bypass at 28 degrees C with the left ventricle vented. An initial 5-minute coronary perfusion was followed by 2 minutes of perfusion every 15 minutes for the cardioplegic solution (8 degrees C) and every 30 minutes for 3 minutes with cold blood or cold blood with potassium (8 degrees C). Hearts receiving cold blood or cold blood with potassium had topical cardiac hypothermia with crushed ice. Peak systolic pressure, rate of rise of left ventricular pressure, maximum velocity of the contractile element, pressure volume curves, coronary flow, coronary flow distribution, and myocardial uptake of oxygen, lactate, and pyruvate were measured prior to ischemia and 30 minutes after restoration of coronary flow. Myocardial creatine phosphate (CP), adenosine triphosphate (ATP), and adenosine diphosphate (ADP) were determined at the end of ischemia and after recovery. Changes in coronary flow, coronary flow distribution, and myocardial uptake of oxygen and pyruvate were not significant. Peak systolic pressure and lactate uptake declined significantly for hearts perfused with cold blood but not those with cold blood with potassium. ATP and ADP were lowest in hearts perfused with cardioplegic solution, and CP and ATP did not return to control in any group. Heart water increased with the use of cold blood and cardioplegic solution. Myocardial protection with cold blood with potassium and topical hypothermia has some advantages over cold blood and cardioplegic solution.

Structural changes and impairment of function associated with freezing and thawing in muscle, nerve, and leucocytes

Abstract In some tissues, especially of skeletal muscle, leucocytes, and peripheral nerves, the effects of freezing to low temperatures, e.g., −78 or −150 °C, and thawing have been a total loss of function and a severe cellular structural disturbance. In the case of muscle, the fibers are converted during thawing into a highly condensed form, called thaw-rigor, in which the individual sarcomeres are shortened beyond a supercontracted state. This effect must be avoided if muscle is to be frozen and stored successfully. Leucocytes and especially the neutrophils are seriously affected by exposure to low temperature. The result is a general swelling of the cell and of its nucleus which is suggestive of damage to the membranes of the cell. Rat cutaneous nerves frozen and exposed to temperature below −15 °C showed after thawing a general disturbance of the myelin involving rupture and separaration of the lamellae. In the axoplasm the neurofilaments and neurotuabules are converted into small aggregates and the mitochondria appear to be swollen. Nerves frozen at −5 and some at −10 °C showed little change in structure, the myelin and axoplasm exhibiting a normal appearance. A check on viability by excitation from an electric stimulus indicated survival after exposure to −5 and in a few cases to −10 °C. A study of freeze substituted preparations established the presence of intracellular ice when the tissues were frozen rapidly to temperatures below −15 °C. Preliminary investigation of the effect of cryoprotective agents, glycerol and dimethyl sulfoxide, on cutaneous nerves shows in electron micrographs that the axoplasm and the Schwann cell cytoplasm are shrunken during direct exposure to 15% solutions.

Cold Blood–Diltiazem Cardioplegia

The calcium channel blocker, diltiazem, has been studied in the same model used for evaluation of cold blood-potassium cardioplegia. Six dogs (Group 1) had one hour of myocardial ischemia with topical ice (myocardial temperature, 7 degrees +/- 2 degrees C) after coronary perfusion with 200 ml of cold blood (5 degrees +/- 1 degree C) containing diltiazem, 400 micrograms per kilogram of body weight. Seven dogs (Group 2) had two hours of ischemia after perfusion with 200 ml of cold blood containing 200 micrograms/kg and reperfusion every 30 minutes with 100 ml of cold blood and diltiazem, 100 micrograms/kg. Baseline studies were repeated after rewarming and 40 minutes of reperfusion. No inotropic agents or calcium were used. Heart rate, peak systolic pressure, velocity of the contractile element, peak + rate of rise of left ventricular pressure (dP/dt), peak - dP/dt, dP/dt over common peak isovolumic pressure, left ventricular compliance and stiffness, and heart water were unchanged in Group 1. In Group 2, heart rate slowed (p less than 0.025) and compliance decreased (p less than 0.02). In both groups, coronary vascular resistance declined (p less than 0.001) and recovery of adenosine triphosphate (p less than 0.001), adenosine diphosphate (p less than 0.025), and the adenosine pool (p less than 0.001) was impaired. Ultrastructure was well preserved, but myofibrillar lesions were noted in Group 2. Diltiazem cardioplegia was associated with good functional recovery, but there was impairment of high-energy phosphate metabolism.

Survival, Respiration and Morphology of Sarcoma-37 Cells Exposed to Liquid Nitrogen.

Summary 1. The survival of mouse sar-coma-37 cells frozen in liquid air has been quantitatively estimated by determinations of the rate of cellular respiration. The results were checked by morphological examination of the frozen incubated tissue and by in vivo growth. 2. Measurements of the rate of respiration were reliable as an index of survival if the cells were incubated for more than 2 hours at 37°C following freezing, and if they were suspended in a physiological medium compatible with normal survival and recovery of injured cells. 3. Krebs-Ringer-Phosphate medium was unsuitable, but equal parts of partially neutralized horse serum and Krebs-Ringer-Phosphate provided an adequate medium. 4. The respiration and survival of sarcoma-37 cells, pretreated for 2 1/4 to 3 minutes in the ethylene glycol in Krebs-Ringer solution then frozen in liquid air was 50 to 60% of the unfrozen controls. 5. The respiration of untreated frozen sarcoma-37 was not usually significant. Morphological analyses of these tissues indicated fewer than 1% surviving cells in most experiments. 6. No significant difference in survival was observed between the rapidly and slowly frozen tissues. 7. Good correlation was observed between the morphological and respiratory characteristics of the frozen incubated tumor cells.

Distribution of myocardial blood flow during extracorporeal circulation

Abstract Distribution of myocardial blood flow was studied during extracorporeal circulation in normal dog hearts. Clinical modalities frequently used to facilitate technical maneuvers were evaluated for their effects on distribution of blood flow and compared to ultrastructural changes. Results do not indicate that changes in distribution alone are responsible for subendocardial ischemia. Anoxia and resultant edema are more important. Protection is provided by topical hypothermia.

Topical Cardiac Hypothermia: The Effect of Methylprednisolone Sodium Succinate

We evaluated the effects of methylprednisolone sodium succinate (MPSS) on 60 minutes of myocardial ischemia during profound (5 degrees C) topical cardiac hypothermia (ice chips) in a canine right heart bypass preparation. The ventricular function curve shifted to the right and downward, but not significantly, after ischemia, and stroke work declined significantly for both control and treated dogs. Contractility (rate of rise of left ventricular pressure and maximum velocity of the contractile element) declined for both groups but not significantly. Total coronary flow, oxygen consumption, and metabolism of lactate and pyruvate were not different for control and treated dogs. Ultrastructure of the outer and inner myocardium did not demonstrate benefit from MPSS. Intracellular and extracellular edema of moderate severity was slightly worse in the subendocardium, and reversible mitochondrial injury of a mild to moderate degreee was symmetrically present. Ice-related injury was not noted. We were unable to deomonstrate that pretreatment with MPSS favorably alters cardiodynamics or ultrastructure after 60 minutes of profound topical cardiac hypothermia.

Effect of cryoprotective agents on rat cutaneous nerves

Abstract Desheathed rat cutaneous nerves were exposed to various concentrations of ethylene glycol (EG), glycerol and dimethyl sulfoxide (DMSO) at temperatures of 1, 24, and 38 °C for periods of time ranging from 5 to 60 min. Measurements of the percent recovery of the original action potential (AP) were determined after removal of the cryoprotective agent (CPA) under various conditions, i.e., temperature, time and sequence of rinsing. A comparison of the results obtained after the nerves were exposed directly to a 15% concentration of the three CPAs at 1 °C for a 15-min period showed that the percentage of recovery of the AP was 90, 69, and 36% of the original values when treated with DMSO, EG, or glycerol, respectively. In all three groups, the nerves were rinsed at 1 °C for 15 min. If the exposure to glycerol at 1 °C was carried out in a gradual stepwise manner, the recovery of the AP in 10 and 15% solutions ranged from 58 to 64%. If the temperatures of the exposure and rinse were increased to 24 and 38 °C, glycerol produced some toxicity within 10 min and after 25 min no recovery of AP was obtained. The results of a 10-min direct exposure to EG at 1 °C showed a moderate decrease in recovery of the AP as the concentration was increased from 10 to 15–20%. Increasing the exposure time to 15 and 30 min at 1 °C also contributed to further reduction in recovery. DMSO, however, in concentrations of 10, 15, and 20% produced only a slight decline of AP after a 5–15 min exposure at 1 °C. Recovery ranged from 96% after 10 min in a 10% solution to 88% after 15 min in a 20% solution. Toxicity became more apparent with DMSO when nerves were exposed to 30% concentrations for 5–10 min; the latter time resulted in a 49% recovery of the AP. Exposure of nerves to a CPA solution containing isotonic concentrations of electrolytes resulted in a 10–30% improvement in recovery when compared with specimens treated with lower levels of salt. The effect of raising the temperature of the rinse to 38 °C and increasing the wash time to 20 min was studied in a few selected experiments. After a direct 15-min exposure to a 15% solution of a CPA at 1 °C the recovery in the case of glycerol was significantly increased with such treatment whereas with EG and DMSO it remained unchanged. There was no evidence of thermal or cold shock in this work.

Ultrastructural observations in canine kidneys perfused hypothermically for 7 days in a comparative study of three preservative solutions

Abstract To determine the effectiveness of different renal perfusates, a comparison was made of the ultrastructure of canine kidneys perfused hypothermically for 7 days with (i) cryoprecipitated plasma (CPP); (ii) plasma protein fraction (PPF); or (iii) canine serum (CS). Each perfusate contained the reducing agents ascorbic acid and glutathione, and the redox level of the perfusate was maintained by potentiostatic control. Ultrastructural analysis was limited to the renal cortex from needle biopsies taken prior to perfusion, after perfusion, and 1-hr post-transplant. All kidneys failed soon after transplantation—turning dark, apparently from vascular blockage. In the CPP-perfused group some fibrin clotting was observed along with minor glomerular changes. The endothelium appeared continuous but beaded, and the epithelium showed retracted foot processes and/or villous extensions. The tubular areas displayed good cellular preservation. The post-transplant biopsies appeared similar, showing no further fibrin deposition but extensive erythrocyte blockage. In the PPF group, serious glomerular changes were observed after perfusion. Both the endothelium and epithelium lacked characteristic profile, and cytoplasmic inclusions were reduced. A flocculent precipitate was found in both the capillary lumen and the urinary space. The tubular regions in this group appeared to be well preserved. After transplantation, the PPF group showed progressive deterioration in both the renal corpuscle and tubules. Kidneys perfused with CS had some glomerular endothelial injury, but the epithelial cell appeared nearly undisturbed. In the tubular regions, the proximal and distal convoluted portions seemed well preserved but the intertubular capillary endothelium showed discontinuity. Upon transplantation, the ultrastructure was essentially similar. In all 1-hr transplant biopsies there was evidence of erythrocyte aggregation but no indication of fibrin or platelet clotting. From the ultrastructural observations made, it appears that significant endothelial injury did occur in the PPF and CS groups during the course of perfusion whereas little or no change was found in the CPP group.