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Anoxia in the newborn rat 1954

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ANOXIA IN THE NEWBORN RAT by SYDNEY SEGAL A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF ARTS in the Department of Physiology We accept this thesis as conforming to the standard required from candidates for the degree of MASTER OF ARTS. Members of the Department of 1 Physiology • « o • • » s e « e f t e i t e t e t i l t t Y « « o • » t> e * o * « • » e » 8 » e 4 * THE UNIVERSITY OF BRITISH COLUMBIA Ap r i l , 1954 ABSTRACT of thesis submitted by S. Segal in partial fulfilment of the require- ments of the degree of Master of Arts in the Department of Physiology, Univer- sity of British Columbia, April, 1954. ANOXIA IN THE NEWBORN RAT A technique was developed for studying the effect of anoxia in the newborn rat with particular reference to persistence of electrical activity i n the heart. In contrast to previous investigations in this f i e l d , no drastic surgical procedures were used, and the animals were held i n a relatively undis- turbed state in a closed temperature regulated chamber which could be f i l l e d with a gas mixture of any desired composition. Eleven newborn rats could be placed in the chamber at the same time under the same conditions, and electrocardio- graphic recordings could be obtained simultaneously from four animals at a time. Anoxia was produced by flushing and f i l l i n g the chamber with tank nitrogen (99.9$ N2), and the period of persistence of electrocardiographic ac- tiv i t y was determined taking as endpoint the last recorded electrical potential from the heart. Two hundred and thirty-two rats were used, ranging i n age from three hours to eight days postnatal. The results obtained agree substantially with those reported by other workers using cruder methods. The "survival time" of electrocardiographic activity in the four day old group was only half that observed in newborn rats less than twelve hours postnatal, the difference being highly significant. However, there was no further significant change i n "survi- val, time" during the period from four to eight days. The technique developed should prove useful in studying many problems of neonatal physiology. i TABLE OF CONTENTS Page I. Introduction 1 A. Problem of Anoxia in the Newborn 1 Bo Experimental Studies on Anoxia of the Newborn 2 1. Historical Background . 2 2. Effect of Age ... 3 3. Effect of Maturity of the Species at Birth 3 4. Effect of Temperature 5 5. Effect of Carbohydrate Availability on Survival 5 6. Effects of Nutritional Status, Hydration, and other Effects 6 7. Observation of the Gasping Pattern in the Isolated Head 6 8. Metabolic Aspect of Anoxia in the Neonate 8 9. Methods Used in Experimental Studies: an Evaluation 11 C. Plan of Present Investigation 14 II. Methods 15 A. Animals 15 B. Measurement of Electrical Activity of the Heart 15 C. Production of Anoxia 17 D. Temperature Control 18 E. Procedure 18 III. Observations and Results 21 IV. Discussion 23 V. Summary ....... 25 VI. Acknowledgements 26 VII. Bibliography . . . 2 7 i i LIST OF FIGURES Page Figure 1. Infant Mortality Rates by Age following 1 Figure 2. Survival Time of Gasping Reflex, Related to age., following 3 Figure 3. Pattern of Carbohydrate Metabolism • 9 Figure 4. Arrangement of Subjects in Nitrogen Chamber following 16 Figure 5. Detail of Electrodes and Immobilization of Subjects following 16 Figure 6. Electrocardiographic Records of Neonatal Rats ... following 17 Figure 7. Recorder Connected.to Electrodes Housed i n Airtight Plastic Box following 17 Figure 8. Airtight Chamber, Showing Path of Nitrogen Flow., following 18 Figure 9. Scatter Diagram, Showing Individual Data Relative to Persistence of Electrocardiographic Activity ..following 21 Figure 10. Chart, Showing Persistence of Electrocardiographic Activity, According to Age Group following 21 LIST OF TABLES I. Relative Persistence of gasping Activity in New- 4 borns of Various Species in Pure Nitrogen ... I I . Duration of Patterns of Gasping in Heads of Neo- natal Mice Breathing Pure Nitrogen 6 Table I I I . Synopsis of Data Obtained i n Review of Experiments with Specific Enzyme Poisons on Neonatal Rats Breathing Air and Pure Nitrogen, Respectively 10 Table IV. Persistence of Electrocardiographic Activity in Two Hundred and Thirty-Two Anoxic Neonatal Rats 22 Table V. Persistence of Certain Activities i n Anoxic Neonatal Animals 24 Table Table I. I N T R O D U C T I O N 1 A. PROBLEM OF ANOXIA IN THE NEWBORN According to Yandell Henderson (2l), the f i r s t fifteen minutes consti- tute the most dangerous period of l i f e ; more lives are lost i n that interval than in any subsequent month* Of 11, 147 births- at Guy's Hospital from 1946 to 1949, Gibbard (18) has calculated that for every 1,000 births, 107 died of anoxia alone. Morison (36) observed that of 397 live born infants who died i n their f i r s t three days, one- half succumbed to anoxia caused by some maternal or obstetrical abnormality, with no sign of congenital anomaly, trauma, infection or any non-respiratory lesion. In a review of the 33 year period from 1915 to 1948, Wegman (52) demonstrated the significant reduction of mortality rates from a l l causes i n indivi- dual age groups of the human population. This trend included the under-a-year and the under-a-month categories. In contrast, there was no apparent improvement during this period i n the mortality figures for newborn infants during the f i r s t twenty-four hours of l i f e (Fig. l ) . Anoxia may occur i n utero. resulting i n s t i l l b i r t h or i n death during the f i r s t few hours. Interference with the baby's supply of oxygen may also occur during delivery or i n the early neonatal period, and may result in irreparable injury or death. As Haldane stated more than thirty years ago: "Anoxemia not only stops the machine but wrecks the machinery." (20). The so-called "devastation areas" of the cerebral cortex, described by Courville (8) and by Schrieber (38) stand as mute evidence of the end effect of a period of oxygen deprivation. There are ample opportunities for interference with oxygen supply during intra-uterine l i f e , for the gas must be transported through devious path- ways and across several membrane barriers before i t reaches the foetal brain. Fig. 1. Infant Mortality Rates by Age. showing apparent lack of improvement i n the age group under one day (52). To follow page 1. Certain regions are particularly critical, especially the placenta and umbilical cord. The oxygen supply remains precarious during the transition of birth. Once the infant is delivered and the umbilical cord is tied, pulmonary respiration must immediately take up the function recently served by the placenta. This is dependent on impulses arising in the respiratory centers in the brain—depression of these centers will often delay the onset of breathing and may result in fatal anoxia. LeLong (30) considers that anoxia is responsible for the great majority of deaths in utero before and during parturition and is also responsible, directly or indirectly, for a large part of the mortality and morbidity immediately after birth. Pre-natal and post-natal anoxia cannot be considered separately, for the latter is usually the result of circumstances originating during or even before birth. B. EXPERIMENTAL STUDISS ON ANOXIA OF THE NEWBORN 1. Historical Background Probably one of the earliest reports in this field was that of Le GaLlois (29) in 1813. He observed that the newborn rabbit had an extraordinary ability to survive without oxygen, as measured by the duration of respiratory movements after submersion, decapitation, opening the thorax and extirpation of the heart. In the same century, Paul Bert (5) described an unusual persistence of respiratory movements in newborn rats and cats, when submerged in water. In subsequent investigations by Avery and Johlin (2), by Selle and Witten (44) and by Fazekas et al. (14) , their subjects included mice, rats, rabbits, cats, dogs and guinea pigs of various ages. Some of these were rendered anoxic by breathing nitrogen, carbon dioxide, argon, hydrogen or illuminating gas. In others, treatment consisted of ligation of the cerebral vessels, decapitation or injection of sodium cyanide. Duration of mandibular movements decreased progressively with advancing age. In neonatal mice made anoxic by breathing oxygen-free gases ( 2 ) , these gasping movements lasted three to six times as long as i n adults under the same conditions. Pazekas et a l . (14) suggested that such apparent prolongation of l i f e was due to the poikLlothermic cooling of the neonate, which acted as a power- ful factor in reducing cerebral metabolic requirements. 2. Effect of Age Glass et a l . (l8) examined the possibility of permanent injury to rabbits as a result of such^prolonged anoxic episodes. Prematurely delivered foetuses at the gestational ages of twenty-nine to thirty-one days survived, on the average, forty-four to thirty-four minutes, respectively, i n pure nitrogen. No permanent effects were observed after such^exposure and the animals developed into apparently normal adults. Newborn rats, left under water at 37° C. for forty minutes and then removed, recovered completely ( 14) , and developed to apparently normal adulthood. In the rabbit experiments (18), i t was found that the respiratory move- ments of prematurely delivered rabbits persisted for a longer time than those of full-term newborns. Furthermore, i t was shown that by delaying parturition with the use of hormonal therapy, the post-mature subjects exhibited a persistence of gasping activity that corresponded to neonates whose age equalled the period of post-maturity. Thus parturition i t s e l f was merely incidental to the relationship of age to this type of survival time. Similarly, newborn rats continued to gasp for f i f t y minutes during oxygen deprivation; at one day of age their limit was reduced to forty minutes, while the adult stopped after two minutes. Such a re- lationship in the isolated heads of mice i s represented graphically i n Fig. 2 from data published by Thorns and Hiestand ( 4 9 ) . 3 , Effect of Maturity of the Species at Birth Newborns of several species were subjected to pure nitrogen breathing SURVIVAL TIME OF GASPING REFLEX, RELATED TO AGE. I9..J..20+ (Thoms a Hiestand) Fig. 2. Survival Time of Gasping Reflex, Related to Age, showing the decreasing duration of mandibular movement in the isolated head of the mouse, with advancing age. The survival of the f i r s t phase of gasping, which persists to adult l i f e , is shown at an almost constant value. To follow page 3. \ 4 . at an environmental temperature of 24° C. Fazekas et a l . (14) found that the persistence of their respiratory movements, shown i n Table I, was related to the degree of maturity of the particular species at this age. TABLE I - RELATIVE PERSISTENCE OF GASPING ACTIVITY IN NEWBORNS OF VARIOUS SPECIES IN PURE NITROGEN. Species Average persistence of Gasping movements (min.) Newborn rats 50 Newborn cats 25 Newborn dogs 23 Newborn rabbits 17 Newborn guinea pigs 7 (Adults, any species 3) Another comparative study by Glass et a l . (l8) yielded results of a similar order, although the endpoint was defined somewhat differently. One group of investigators (l8) recorded the time of the last spontaneous gasp, while the other (14) fixed the end point as the time at which "movements could no longer be evoked by any stimulation". In both of these experiments, the most prolonged gasping activity during anoxia was associated with the species that was characteristically the least mature at birth. Thus, the newborn rat had no hair or teeth. Its eyes were not yet open. It was totally dependent upon i t s mother and acted like a bulbospinal animal. The newborn guinea pig, on the other hand, which gasped for only seven minutes, was relatively mature, with co-ordinated locomotion, righting reflexes, temperature regulation and, therefore,with a functioning cephalad portion of the brain stem ( 2 4 ) . 5. 4 . Effect of Temperature. Himwich et a l . (25) varied the ambient temperature in which one-day- old rats were exposed to an atmosphere of pure nitrogen. When temperature was increased from 24° C. to 34° C, the duration of gasping activity was reduced from f i f t y minutes to twenty-one minutes. Adult rats did not appear to be affected i n this manner by temperature change, presumably because of the homeothermic nature of the mature animal. Assuming that glycogenolysis was the means by which energy was released under anaerobic conditions, Dixon (lO) suggested that elevation of temperature probably resulted i n an increase i n the demand for substrate in the brain, but that this increase exceeded any acceleration of the rate of cerebral glycogenolysis. Miller (35) exposed guinea pigs less than one day old to an atmosphere of 5$ CO2 and 95$ N2, and observed that animals at room temperature were a l l dead-, after four and a half minutes, while most of those which had been cooled survived this period of anoxia. 5. Effect of Carbohydrate availability on Survival Himwich (26, 27) observed that administration of insulin to newborn rats prior to exposure to anoxia reduced to half the period during which gasping activity persisted. On the other hand, prior injection of glucose to rats eight to ten days old significantly increased the period of gasping activity. Selle (43) obtained similar results with respect to the persistence of gasping movements of the mandible following decapitation. He also found that injection of glucose along with insulin nullified the deleterious effect of the latter. Hiestand et a l . (23) used the same technique of observing movements of the mandible i n the decapitate head, and found that administration of epinephrine and pituitary extracts increased the survival period, possibly because of their hyperglycemic effect, while a period v o of fasting (twenty-two hours) reduced survival. 6. Effects of Nutritional Status. Hydration, and other effects. Hiestand et a l . (22, 23) have reported that starvation reduced the resistance to hypoxia in direct relation to the duration of the period of inanition, confirming earlier reports of Selle (4l0» However, they observed no such effect of dehydration, and the period of gasping was actually maximal in mice which had lost twenty per cent.of their body weight due to water loss while restricted to a dry diet. Selle (4-1, 42) has reported differences i n survival related to differences i n individual l i t t e r s . Britton and Kline (;7l) found the adult female rat more resistant to anoxia than the male, but they were unable to demonstrate such a difference due to sex at the neonatal level. 7. Observation of the Gasping Pattern in the Isolated Head. In these experiments, decapitation was performed with a razor just caudad to the fore-limbs, leaving the chemoreceptors and medullary centers intact. Mandibular movements i n the isolated head were used as an indication of active gasping related to activity of the respiratory center. TABLE II DURATION AND PATTERNS OF GASPING IN HEADS OF NEONATAL MICE BREATHING PURE NITROGEN. Age (days) No. of Mice Duration of First Series Total Survival Time, both series. (sec.) + S.E. (sec.) + S.E. 1 8 32.2 2.2 1652.1 135.4 3 6 19.0 1.5 1459.2 101.3 8 10 19.5 2.5 574.3 127.7 12 11 13.3 1.1 71.8 10.3 19 13 18.6 1.8 19.9 1.8 20+ 49 16.05 0.25 16.05 0.25 Data from Thorns and Hiestand (49) are summarized i n Table I I . In the isolated heads of white mice, the survival time of mandibular movements was related inversely to age. The curve was relatively steep for the f i r s t eight to twelve days reaching the adult value at the age of nineteen days and then levelling off. The gasps occurred characteristically i n two distinct volleys punctuated by a period of inactivity. In this report, the f i r s t series of volleys lasted from a quarter to a half a minute. The second series persisted up to about half an hour at the age of one day but decreased with age to become barely discernible in the adult mouse. Hiestand et a l . (23) have related this pattern of two periods of gasping separated by an apneic pause, to the different levels of integration of the respiratory centers. The primitive center, being more rugged, might be expected to survive longer under anoxia. They suggested, therefore, that the pro- longed second series of gasping was probably due to activity of the primitive center. As evidence for their theory, they pointed out that these gasps were of a slow, rhythmic nature, and definitely apneustic i n character. The gasps in the f i r s t series, however, were more rapid and less apneustic, and were probably caused by the superimposed regulation of the higher centers. \ Using the heads of rats younger than six weeks, Selle and Witten (45) obtained results comparable with those of Thorns and Hiestand (49). The f i r s t series of gasps ceased after less than a minute. After an interval of thirty to f i f t y seconds, the second burst of gasping movements occurred and lasted from twenty to forty minutes, the period diminishing with increasing age, and disappearing entirely at the age of six weeks. Moreover, the effect of temperature, already described, affected the duration of the second series of mandibular movements. The f i r s t series has been identified with aerobic metabolism, and the second with anaerobiosis. ( 2 4 ) . - Selle (42, 43) has found that insulin or glucose, administered to the neo- natal rat prior to decapitation, exerted practically no effect upon the f i r s t , or aerobic series of gasping movements: these agents affected only the second, or an- aerobic phase. When the dose of insulin was sufficiently large, there was complete disappearance of this second phase. Likewise, the effects of pituitary and adrenal hormone administration, discussed in connection with availability of carbohydrate, were confined to the anaerobic gasping time0 None of these agents had any apparent effect on the total gasping time i n the adult animal, which does not show this second anaerobic period of gasping. The injection of Iodacetate, an enzyme toxin hat blocks the anaerobic glycogenolytic process, did not affect the f i r s t phase but eliminated the second gasping period, the'pattern resembling that of the insulin-treated neonate or the untreated adult, ( ? 3 , 42). On the other hand, sodium cyanide, which i s known to i n - activate the cytochrome oxidase system and thus prevent oxygen utilization by the tissues, appeared to block the f i r s t aerobic phase but had no effect upon the second phase (42). 8. Metabolic Aspect of Anoxia i n the Neonate. Studies with specific enzyme toxins, as reviewed i n Table III, have shown that the metabolic processes responsible for this added resistance to anoxia involved the established patterns of carbohydrate metabolism (Fig. 3 ) . The salient factor i n the neonate,; appears to be increased activity i n the glucose-to-lactate portion of the metabolic pathway for carbohydrate. Himwich (24) accounted for this specific difference on the basis of enzymes and the variation of their respective concentrations with developmental changes from neonatal to adult l i f e . Apparently, with this localized increase i n activity, the neonate utilizes less oxygen and more glucose, and produces more lactic acid, even under fully aerobic conditions (A, I S , 50, SI, :53). In anoxia, further increase i n the rates of carbo- hydrate consumption and lactate production have been demonstrated (11', 12, 13, 53). Within the neonatal period, the effect of age on resistance to anoxia was reflected i n progressive reduction i n this high rate of lactic acid production (26). According to Himwich (24), survival of the central nervous system, considered to be the area most vulnerable to the effects of anoxia ('fil), depends upon the metabo- lism of glycogen stores i n the brain* Brinkman (j6.) however, related the anaerobiosis rather than brain storage, to.blood glucose levels. Consequently, there has been speculation as to the possible function of the abundant glycogen deposits in the lungs of foetuses and neonates, as reported recently by LeLong and Laumonier (31). PATTERN OP CARBOHYDRATE METABOLISM (24) GLYCOGEN GLUCOSE' 'HOSPHATE \ Warburg-Christian Dickens (aerobic) RIBOSE ETC. Embden Meyerhof {, anaerobic) TRI (anaerobic) LACTIC ACID OSE PHOSPHATE (inhibited by iodacetate) PHOSPHOGLYCERIC ACID _• - (inhibited by fluoride) PYRUVIC ACID (inhibited by - cyanide) Krebs (aerobic) C02 + H20 FIGURE 3. PATTERN OF CARBOHYDRATE METABOLISM (24) TABLE I I I SYNOPSIS OP DATA OBTAINED IN REVIEW OF EXPERIMENTS WITH SPECIFIC ENZYME POISONS ON NEONATAL RATS BREATHING AIR AND PURE NITROGEN, RESPECTIVELY (14;, 26;, 28, 34). Toxin injected None CN Fluoride Iodoacetate Gas respired » i Air N2 Air 1 Air N 2 Air N 2 Metabolic cycles permitted by gas and toxin combined: Warburg-Christian-Dickens + 0 0 + 0 + 0 Embden Meyerhof + + + *± *± -Krebs Citric Acid + 0 0 End product postulated CO2 + H2O Lactate Lactate Phos. gly.c. Phos. glyc. Triose ph. Triose ph. Lactate accumulation + . +++ +++ 7 ? + + Duration of gasping (min.) Continues 50 50 50 , 16 50 3 LEGEND: Cycles: + complete cycle: ^ + cycle arrested part way; + cycle shortened considerably! 0 cycle inhibited completely. Product: Phos. g l y c ; phosphoglycerLc acid; Triose ph.; triose phosphate. Lactate: + normal concentration; +++ greatly increased concentration. l l o These authors made use of some of the available metabolic evidence to explain the successful revival of a baby who had been delivered by Caesarean section fifteen to twenty minutes after maternal death. They suggested that this tolerance of anoxia i n the human neonate may be related to: i (a) the undeveloped state of the cerebral cortex, leading to a decreased metabolic rate and to decreased vulnerability; (b) lowering of body temperature, which would decrease the cerebral metabolic rate and i t s vulnerability; ^ (c) capacity for anaerobiosis, as shown in the rat experiments. 9. Methods Used i n Experimental Studies: an Evaluation a) Respiratory Movement o In the published reports dealing with resistance of the neonate to anoxia, the time of the last gasp has been the usual endpoint. In the intact animal, there have been two types of observations: (i) the last spontaneous gasp and ( i i ) the last gasp i n response to a stimulus. Considering" the last spontaneous gasp, the data obtained must have lacked a reasonable degree of precision, since the gasping movements i n advanced anoxia occurred after progressively prolonged and irregular intervals, each gasp being separated from the previous one by as many as three to five or more minutes (39). An observer attempting to watch a whole l i t t e r of eight to fifteen subjects at one time, might miss the very-last one or two quick gasps of any one animal while looking at another and would record the so-called survival time with an error of ten minutes or more. Unintended disturbance of the animal may also stimulate a gasp, so that i t i s difficult to obtain uniform results. Where the endpoint was taken to be the last gasp elicited by stimulation, there i s no indication either of the exact nature of the stimulus or the means taken to obtain reproducible results. In addition, experience with use of electric 12. shock (39) would suggest that a stimulus of sufficient intensity and duration to provoke a gasp i n a neonatal anoxic rat might also have other side effects. Recently, Mayer (33) reported a number of newborns which had not been observed to breathe until after the f i f t h minute. Their condition was described as a "false state of anoxia" because radiography showed adequate pulmonary expansion and there was apparently adequate respiratory exchange, as a result of slight substernal undulations. (From the biometric viewpoint, i t was noted that with only two exceptions, the vast number of reports failed to provide any indication of the reproducibility of results, or of the extent of variation of the data from the published arithmetic means.) (b) Blood Gas Studies When newborn puppies were subjected to an atmosphere of pure nitrogen, Himwich et a l . (25) observed that, after only five minutes, there was no measurable amount of oxygen in the arterial blood, although respiratory movements continued for another twenty or thirty minutes. Smith and Kaplan (46, '47) found no correla- tion between blood oxygen level and the period of delay before the f i r s t spontaneous breath, nor between the oxygen level at birth and i t s rate of subsequent change. Even with an adequate arterial oxygen saturation, (e.g., ninety-three per cent) prematures were observed by Graham et a l . (19) to be breathing irregularly as though suffering from hypoxia. Carbon dioxide studies have been equally equivocal. Eastman (13) found that, in the normal newborn, the range of carbon dioxide tensions (38-60 mm Hg PC02) was extremely broad with no obvious relationship to respiratory activity. (c) neurological Signs The system i n which reversibility of the effects of neonatal anoxia appears to be c r i t i c a l i s the central nervous system (61, 24). 13. Selle and Witten (44, 45) observed the pupillary responses i n the isolated head and also the trunk reflexes i n the spinal animal after decapitation. The former were stimulated by means of a bright light, the latter by the applica- tion of an allegedly painful electric shock, the stimulating electrodes being applied to one limb and to the cutaneous surface of the abdomen. Even i f the tests had been performed on subjects i n a more physiological state than that of decapitation, these techniques of stimulation would not be very reproducible when applied to small animals. Furthermore, there appears to be a basic fallacy with any interpretation of reflex activity under these circumstances, since an infant suffering from severe asphyxia neonatorum may have absent reflexes and s t i l l pro- ceed to spontaneous or aided complete recovery (16, 39). Electroencephalography would seem to have merit i n this regard. How- ever, there are two considerations that would preclude i t s application to this problem: (a) In depressed states, such as in heavy narcosis or after a convulsive episode, there may be a temporary but complete lapse of a l l electrical activity from the brain. This so-called silent period could result in a falser interpreta- tion, (b) In the failing central nervous system of a moribund subject, the rate of decrease in the amplitude of electrical activity i s so gradual and the voltages may be so minute to begin with, that the electroencephalographic potentials would slowly become indistinguishable from electrical artefacts and there would be no distinct endpoint (l5). (d) Cardiovascular Signs In the peripheral circulation, the determination of blood pressure i n ( neonates, particularly of small species, i s both time consuming and inaccurate, with available techniques. Swann (48) reported that he was unable to measure the pressures accurately in neonatal pups or to correlate the results with chances of surviving an anoxic episode. 14. Selle and Witten (40, 44, 45) and Himwich et a l , (25) have observed persistence of cardiac activity in neonates after the onset of apnea. Indeed, in severely anoxic newborn human infants, the heart may continue to beat long after the baby i s no longer capable of responding to resuscitatory efforts. However, when enzyme toxins were administered (26), there was abrupt termination of both gasping and cardiac activity, Selle (40) opened the thorax and observed the continued beating of the heart after gasping had ceased or after decapitation, and noted the time at which cardiac activity ceased. However, there are some serious objections to such drastic procedures. On the other hand, determination of electrical activity in the heart by electrocardiography can be carried out on the intact neonate with very l i t t l e disturbance. Although electrocardiographs have been obtained by a number of investigators using foetuses, neonates and adult small animals, i t does not appear to have been used as an index of cessation of cardiac activity in anoxic animals, prior to the present investigation. C PLAN OF PRESENT INVESTIGATION The purpose of the present investigation was to study the effect of age on survival of anoxic neonatal rats, using as criterion the persistence of electrical activity in the heart. Cessation of cardiac activity i s the common clin i c a l criterion of death, and has been used by Selle (40) i n assessing the survival of newborn rats i n an atmosphere of nitrogen and following decapitation. He opened the chest and observed the beating of the heart directly. In the: present investigations, it'was felt desirable to avoid such drastic measures by recording electrical activity using standard electijcardiographic techniques in an intact animal. To accomplish this, apparatus was developed which permitted electro- cardiographic recordings from up to eleven rats mounted in a closed chamber f i l l e d with nitrogen. I I . M E T H O D S 15. A. ANIMALS The animals used were albino rats of the Wistar strain, obtained from the stock colony of the Department of Animal Husbandry of the University of British Columbia. A few rats of the Sprague-Dawley strain have also been included. The number of rats i n each l i t t e r varied from six to fourteen, but only eleven could be accommodated i n the apparatus at any one time. There was some d i f f i - culty i n determining the exact time of birth when rats were born at night, and i n some cases two hours were required for the delivery of the complete l i t t e r . How- ever, i n view of the wide scatter i n "survival times", i t was fel t that estimation of the birth time within a few hours was sufficiently accurate for the purpose. The rats i n the age range from three to twelve hours have been grouped together, and the average value i s plotted at the average time of seven hours. Those i n the range from twelve to twenty-four hours have been plotted at the average time of twenty-one hours. Subsequent groups are arranged according to the closest day. B. MEASUREMENT OF ELECTRICAL ACTIVITY OF THE HEART Several types of electrodes have been used oh ̂ small animals, each with certain limitations. Richards et a l . (37) embedded needles i n the skin. Agduhr and Stenstrom (l) used small zinc plates, amalgamated with mercury. They wrapped these around the limbs of adult mice, bandaging them with cotton wool dampened with saline. Under ether anaesthesia, the animal was transfixed to a cork plat- form by means of pins driven through the nose, t a i l and each of the limbs. Bauer (3) buried rectangular plates under the skin of young rabbits. However, to save time while working on foetuses, he fastened crocodile clips to their limbs. Lombard (32) made recordings from adults of several small animals, by an entirely different system. She immobilized the anaesthetized subject i n a 16. plastic sling, resting on i t s belly with the limbs hanging downward and dipping into small beakers containing 1 M. zinc chloride solution and zinc electrodes. Various types of amplifying and recording equipment were used by these investigators. In a l l cases, records were obtained from only one animal at a time. In the following experiment, special apparatus was constructed which made i t possible to place up to eleven neonates in a single chamber and record simultaneous electrocardiograph records from four at a time, without i n any way disturbing the animals or the gas mixture. Each animal was held in place by a pair of electrodes which gripped the right foreleg and left hindleg, so that the potentials picked up would correspond to the Standard Limb Lead II of the Electro- cardiograph. The apparatus i s shown in Figure 4. I n i t i a l l y , brass electrode clips were used, but these were replaced later by limb clips, illustrated in Figure 5, which consisted, of two arms of nickel-silver wire, and a c o i l spring which pressed the arms together. The latter electrodes did not corrode, and the tension could be adjusted so that i t was just sufficient to hold the animal securely during the struggling which occurs i n the early stages of anoxia. The electrode connections were shielded, as was the bottom of the ^ plastic box, to reduce the pickup of extraneous potentials. A special selector switch on the panel made possible connection of any desired electrode pair to the recording apparatus. I n i t i a l l y , electrocardiograph records were obtained from each animal i n turn, using a standard clinical model of the Sanborn-Viso— Cardiette, with single channel amplifier and a direct writing thermal stylus. A f u l l minute recording was made from each rat, and i t was found that at least twenty minutes -wMscrequired to obtain records from eleven animals. This gave an c. uncertainty in time of endpoint of at least twenty minutes. Fortunately, i t was Fig. 4. Arrangement of Subjects i n Nitrogen Chamber. The subjects are placed radially, immobilized by the electrodes. The thermometer i s inside the chamber. The switch at lower l e f t connects the electrodes to the amplifier-recorder, four pairs at a time. The switch at right connects an ohmmeter to the subjects, one at a time. To follow page 16. Fig. 5. Detail of Electrodes and Immobilization of Subjects, showing the design of the nickel-silver clips which, attached to the right foreleg and l e f t hindleg of each subject, immobilized the animal and carried to the recorder-amplifier the Standard Limb Lead II of the Electrocardiogram. 17. possible to obtain the loan of an obsolete electroencephalograph machine* which provided a four channel amplifier and ink recorder, and made possible simultaneous observation of E.C.G.Js from four animals at a time* A f u l l run took less than three minutes for a l i t t e r of eight rats, allowing a run of one minute duration on each of the two groups of four animals. For the f u l l number of eleven subjects, a complete observation was possible every five minutes. Typical early and late recordings are shown i n Figure 6. Electrical resistance through each animal: was measured after they were f i r s t attached to the electrodes and again at the end of the experiment. A volt- ohm-milliammeter, used for this purpose, showed that the resistance did not exceed more than 5,000 or 6,000 ohms in any case, although the amplifiers would s t i l l operate efficiently when the resistance was as great as 20,000 ohms. Resistance was reduced to a minimum with an electrode j e l l y . C. PRODUCTION OF ANOXIA The newborn rats, held in place by the electrodes, were surrounded by an air-tight plastic box illustrated i n Figure 7. The edges were sealed with plasticine. Tank nitrogen (testing 99.9$ N2) was humidified by passing through * Amplifier-recorder was manufactured by Electro-Physical Laboratories, Inc., 290 Dyckman Street, New York 34, New York, U.S.A. There was a separate five- stage push-pull amplifier on each of four channels. Each channel had a separate ink-writing galvanometer. Frequency response range was one quarter cycle to . 4,000 cycles. The ink-writing galvanometers had a response that was uniform for 0 to 55 cycles. Paper speed originally was three cm. or six cm. per second but was modified for the present experiment to 1.5 cm. per second, allowing a f u l l minute of recording to be made on a thirty-three inch length of paper. The apparatus was made available through the kindness of the E.E.G. Department, Vancouver General Hospital. ** Modified electrode je l l y as formulated by the Dispensary of the Vancouver General Hospital. Composition: Pulverized Tragacanth No. 1 A.A. 50 Gm. Glycerin 400 Gm. Sodium chloride 400 Gm. Carbolic Acid 10 ml. Pulverized Pumice 90 Gm. Water 1600 ml. ELECTROCARDIOGRAPHIC RECORDS OF NEONATAL RATS LEAD j£. Early RATS Fig. 6. Electrocardiographic Records of Neonatal Rats, showing tracings from four-day-old rats. In the earlier set, the subjects had been anoxic for several minutes. Their heart rate was approximately 40 per minute. In the later set, amplitude was set to a greater degree than i n the earlier group. Activity persisted i n No.6 and No.7 but the endpoint had been reached i n No.5 and No.8. Fig. 7. Recorder Connected to Electrodes Housed i n Airtight Plastic Box, showing 11 subjects i n the nitrogen chamber. The circuits lead through the respective amplifiers and terminate i n the four direct-writing galvanometers, photographed i n the process of tracing electrocardiographic patterns. 18. a water bottle and then flooded through the chamber escaping at the opposite corner through a tube' leading to a water bubbler escape (Figure 8). This pro- vided a water seal, and the slow bubbling indicated the rate of flow of nitrogen and maintained the pressure just slightly above atmospheric. The escaping gas was tested from time to time by a Beckman Model D oxygen analyzer and at no time was the oxygen concentration found to exceed 0.5$. D. TEMPERATURE CONTROL The plastic nitrogen chamber was enclosed in a wooden box 2' x l - ^ ' by 2*, with the top composed of a transparent plate which could be removed. The heat was applied by a sixty watt electric light bulb situated beneath a wooden baffle and was regulated by a Fenwall thermostat. Temperatures inside the nitro- gen chamber were measured with a thermometer and remained within the.range 25.0 + 1.0° C. E. PROCEDURE While the temperature in the chamber was stabilizing and the amplifier was warming up, the baby rats were separated from their mother, weighed, and an identifying number was written on each head. Electrode jelly was placed on the legs which were connected to the electrodes so that the animals were held on their backs with the heads directed towards the center of the panel. The electrical resistance through each animal was then measured and in no case exceeded 6,000.. ohms. The four channels of the amplifier were standardized and calibrated to give a deflection of one centimeter per millivolt. A short trial run was made before the chamber was sealed with plasticine. The nitrogen valve was opened and the time recorded as the starting point of the anoxic period. The apparatus was then placed in the constant temperature box. Fig. 8. Airtight Chamber Showing Path of Nitrogen Flow. The gas follows the circuit indicated by arrows. The under-water escape arrangement is shown at right in a well that was bore out of one of the supporting legs of the apparatus. To follow page 18, J 19. One minute records were run on each rat at intervals of approximately ten minutes. When the amplitude of the deflection corresponding to the QRS complex had diminished to 0.2 centimeters, the amplifier gain on the appropriate channel was increased so that ultimately a point was reached at which the one centimeter deflection corresponded to 0.1 millivolts. The endpoint for any animal was taken as the time of the last recorded electrical activity from the heart. As the end-: point was neared, the frequency of observations was increased to one every five minutes. A small potential error was introduced by adopting this endpoint, for there may have been a final E.C.G. spike during the five minute interval prior to the next recorded and "silent" run, in which no electrical activity was observed. However, since the error was uniform in a l l groups, and was small in comparison with the individual variation i n the data, i t was f e l t that i t should not seriously affect the validity of the results. Its elimination would have required continuous and simultaneous recordings from a l l animals during the entire anoxic period. During the early part of the run, only two or three ECG complexes were recorded on paper during each observation. However, as the endpoint approached, records were made for a f u l l minute. When,finally,two consecutive one minute runs failed to show any recognizable electrocardiographic activity from any member of the l i t t e r , the experiment was discontinued. The calibration of the recorder was rechecked, and the apparatus was then removed from the constant temperature box and a final gas sample taken and analyzed to confirm the absence of oxygen in the chamber. The resistance through each animal ,was also measured to insure that good electrical contact had been maintained throughout the experi- ment. 20. Despite careful shielding and grounding .of connections the broad band corresponding to a 60 cycle induced current occasionally appeared on the record. In this case the interferance was screened out by turning on the f i l t e r circuit contained in the amplifier. Careful attention was also given to any deflections on the record that might have been due to extraneous potentials from neighbouring equipment. These were identified and carefully distinguished from the electrical potentials arising in the heart. ( 21. I I I . O B S E R V A T I O N S AND R E S U L T S Following the onset of anoxia, there was a brief period of hyper- activity lasting three to five minutes, during which there were wide fluctuations i n the electrical base line with clearly recognizable ECG complexes superimposed. This was followed by a period during which there was no perceptible movement of the animal with the exception of occasional gasps, which occurred irregularly and at progressively longer intervals until, finally, no further activity could be observed. After ten minutes, there was no body activity to disturb the electrical base line, with the exception of occasional gasps, and even these disappeared long before the electrocardiographic endpoint was reached. The duration of ECG activity was determined as described above, and the individual data have been plotted as a scatter diagram i n Figure 9, i n which each point represents, the results from a single animal. The mean values for each age group and the standard errors of the means have been calculated according to the * procedure described by Snedecor and are represented i n Table IV and plotted i n Figure 10. The " t " test of significance was applied to comparison of the observed differences. It was observed that the time of persistent electrocardiographic activity i n the f i r s t group (up to 12 hours postnatal) was significantly greater than i n any of the other groups (probability "p" of the difference occurring by chance was less than 1 i n 100 in a l l cases). The time was double that observed in the four day group. On the other hand, no significant difference ("p"^0.05) could be demonstrated between the four day group, and the succeeding age groups. * Snedecor, G.W.: Statistical Methods Applied to Experiments in Agriculture and Biology, Ames, l a . , Collegiate Press, Inc., 1937, pp. 50-55. 250 200- UJ § 150 o < . 100-o d 50 « •s i 0 7 21 HOURS PERSISTENCE OF ELECTROCARDIOGRAPHIC ACTIVITY IN 232 NEONATAL R A T S i R E S U L T S DISTRIBUTED A C C O R D I N G TO A G E • W l S T A R 8TRA IN • S P R A O U E - D A W L E Y • AVERA6E J 3. D « -5 t'4 3 4 5 6 A G E . (DAYS) Fig. 9 . Scatter Diagram, Showing Individual Data Relative to Persistence of Electrocardiographic Activity. Following page 21. P E R S I S T E N C E OF E L E C T R O C A R D I O G R A P H I C A C T I V I T Y IN 232 N E O N A T A L R A T S IN R E L A T I O N T O A G E . 150 C O 3 Z ^ 100 < 50 Q) 5 J T I M E E X P R E S S E O A S T H E E R R O R O F T H E M E A N M E A N 0 i T H E S T A N D A R D NUMBER OF A N I M A L S : S» 82 57 8 8 12 21 1 1 1 1 6 1 8 1—— -18-v. . (•• 2 3 AG E 4 5 6 (DAYS) Fig. 10. Chart, Showing Persistence of FJLectrocardiographie- Activity, According to Age Group. Following page 21. 22. TABLE IV PERSISTENCE OF ELECTROCARDIOGRAPHIC ACTIVITY IN 232 ANOXIC NEONATAL RATS. AGE 1 ' ' 1 GROUP NUMBER IN GROUP MEAN TIME (Minutes) i STANDARD ERROR OF THE MEAN 7 Hours 35 174.7 + 3.6 21 Hours 32 121.1 + 8.1 2 Days , 57 113.6 + 4.1 3 Days 36 124.9 + 2.4 4 Days 12 95.5 + 4.9 j 5 Days 21 87.5 + 5.2 6 Days 16 92.9 + 4.6 7 Days 8 94.2 + 6.8 8 Days 15 87.2 + 8.1 ! 23. ^ I V« D I S C U S S I O N In this study of 232 neonatal rats rendered anoxic by exposure to an atmosphere of tank nitrogen in a closed chamber, the persistence of electrical activity in the heart appeared to decrease with post-natal age, the sharpest decline occurring i n the f i r s t day of l i f e . In Table T. the results obtained i n this series have been compared with data reported by other investigators on the "survival time" in a number of anoxic newborn animals. There i s good agree- ment between the values obtained in this series, using persistence of ECG activity as the criterion of "survival", and those reported by Selle (40) i n which persis- tence of heart beat was observed i n the open chest of a spinal animal. Cardiac activity persists longer than gasping activity i n the whole animal or i n the iso- lated head. Under the same conditions, "survival of activity" i n rat, mouse and rabbit i s similar, while that in the dog and guinea pig i s considerably shorter. This may be related to the more mature condition at birth i n the latter animals, and particularly in the case of the guinea pig, as has been suggested by Pazekas (14) . However, i n a l l species there i s a decrease i n "survival time" following the f i r s t day of l i f e . It may be that this i s the result of a shift i n metabolic pathways occurring during this period of rapid adjustment to extrauterine l i f e . TABLE V- PERSISTENCE OF CERTAIN ACTIVITIES IN ANOXIC NEONATAL ANIMALS Method and Species Post-Natal Age (Days): j 0.5 ± 1 2 4 5 6 7 Gasping activity of the whole animal ( l 8 ) : 1 Guinea Pig 4.5 4.5 ; 4 3.5 3.5 3.5 3 3 Bog 31 17 14 Rabbit 27 20 17 13 11 13 9 7 Mandibular movements of the isolated head: Rat (43) 27 2* 12 Mouse (49) | + S.E. 27.5 + 2.2 24.3 ± 1.7 1̂2' 9.6 ± 2.1 Cardiac activity (exposed heart) (40) : Dog 58 42 31 Rabbit 113 84 58 Rat 103 96 102 99 (electrocardiographic): Rat (present invest." 174.7 121.1 113.6 124.9 95.5 87.5 92.9 94.2 87.2 + S.E. i ± 3.6 . + 8.1 + 4 .1 ± 2.4 ± 4.9 ± 5.2 + 4.6 + 6.8 ± 8.1 25. V. S U M M A R Y A technique was developed for studying the effect of anoxia i n the newborn rat with particular reference to persistence of electrical activity i n the heart. In contrast to previous investigations i n this f i e l d , no drastic surgical procedures were used, and the animals were held in a relatively undis- turbed state in a closed temperature regulated chamber which could be f i l l e d with a gas mixture of any desired composition. Eleven newborn rats could be placed in the chamber at the same time under the same conditions, and electrocardio- graphic recordings could be obtained simultaneously from four animals at a time. Anoxia was produced by flushing and f i l l i n g the chamber with tank nitrogen (99.9$ N 2 ) , and the period of persistence of electrocardiographic ac- tiv i t y was determined taking as endpoint the last recorded electrical potential from the heart. Two Hundred and thirty-two rats were used, ranging i n age from three hours to eight days post-natal. The results obtained agree substantially with those reported by other workers using cruder methods. The "survival time" of electrocardiographic activity i n the four day old group was only half that observed i n newborn rats less than twelve hours postnatal, the difference being highly significant. However, there was no further significant change i n "survi- val time" during the period from four to eight days. The technique developed should prove useful in studying many problems of neonatal physiology. 26. VI. A C K N O W L E D G E M E N T S i The author i s indebted to Professors D. H. Copp, E. C. Black and J. P. McCreary for their guidance and advice during the period of post- graduate training and in the present project, to Mr. A. J. Honour and Mr. H. Hallam for invaluable technical advice and assistance, and to Miss Katherine HoskLn for a l l of the photography and the reproductions. The electroencephalograph was loaned from the EEG Department of the Vancouver General Hospital through the kindness of Dr. N. Auckland. Mr. R. St. L. Ferguson was constantly available to correct maintenance problems and to advise on the detection of artefactual wave forms. The author i s indebted to the National Research Council of Canada for financial assistance provided through a Graduate Medical Research Fellow- ship. 27 VII, B I B L I O G R A P H Y lo AGDUHR, E, and N, Stenstrttm: The Appearance of the Electrocardio- gram i n Heart Lesions'Produced by Cod Liver O i l . Acta Pediatrica 8: 1*93-610, 1928/29o 2. AVERY, R.C. and J . H. Johlin: Relative Susceptibility of Adult and Young Mice to Asphyxiation. Proc. Soc. Exper. B i o l . & Med. 29: II8I1-II86, 1931-32. 3« BAUER, D.J.: The Effect of Asphyxia upon the Heart Rate of - Rabbits at Different Ages. J . Physiol. 93: 90-103, 1938. ht BELL, W.B., Le Cunningham, M. Jowett, H. Mill e t and J . Brooks: The Metabolism and Acidity of the Foetal Tissues and Fluids. B r i t . M. J. 1: 126-131, 1928. 5« BERT, P.: Lsjohe sur l a physiologie de l a respiration, profesees au Museum d'histoire nature He, Paris, J. B. Balliere et f i l s , 1870, quoted by HDMICH, H.E.: Reference (22) of this bibliography. 6. BRINKMAN, R.: Factors Relevant to Foetal and Neonatal Anoxic Tolerance, Anoxia of the^Newborn Infant, A Symposium Organized by the Council for international- Organiza- tions of Medical Sciences, Established under the Joint Auspices of U.N.E.S.C.O. and W.H.O., 123-126, Oxford, Blackwell Scientific Publications, 1953. 7. BRITTGN, S.W. and R. F. Kline: Age, Sex, Carbohydrate, Adrenal Cortex and Other Factors i n Anoxia, Am. J. Physiol. 3Ji5: 190-202, 19U5. 8. COURVILLE, C.B.: Asphyxia as a Consequence-of Nitrous Oxide Anesthesia. Medicine 35s 129-2U5, 1936. 9. 8EEERJ, R.D.G. and T. J , Parkinson: Blood Glucose Changes i n the Newborn. Arch. Dis. Child. 28: 13l*-139> 1953. 10. DIXON, K.C.: The Effect of Rise i n Temperature on the Carbohyd- rate Catabolism of Cerebral Cortex, Biochem. J . 30: Ut83-lli88, 1936. 11. EASTMAN, N.J.: Foetal Blood Studies. I l l The Chemical Nature of Asphyxia Neonatorum and i t s Bearing on Certain Practical Problems, B u l l . Johns Hopkins Hosp. 50: 39-50,1932. f 28 12, EASTMAN, N.J. And C. H. Mclane: Foetal Blood Studies, I I The lactic Acid Content of Umbilical Cord Blood under Various Conditions, B u l l . Johns Hopkins Hosp© UBt 261-268, 1931. 13. EASTMAN, N.J., E, M« K. Gelling and A. M. Delawder: Foetal Blood Studies. IV The Oxygen and Carbon Dioxide Dissocia- tion Curves of Foetal Blood, B u l l a Johns. Hoplins Hosp. 53: 21+6-251+, 1933. 11+. FAZEKAS, J.F., F. A. D. Alexander and H. E. Himwich: Tolerance of the Newborn to Anoxia, Am. J. Physiol. 13l+: 281-287, 191+1. 15. FERGUSON, R.St.L.: Personal communication to the author. 16. FIAGG, P.J.: The Treatment of Postnatal Asphyxia, Am. J . Obst. & Gynec. 21: 537-51+1, 1931. 17. GIBBERD, G.F.: Mechanism, Prevention and Treatment of Asphyxia i n the Newborn Infant. Anoxia of the Newborn Infant, A Symposium Organized by the Council for International Organ- izations of Medical Sciences, Established under the Joint Auspices of U.N.E.S.C.O. and W.H.O., 26-1+1, Oxford, Black- well Scientific Publications, 1953. 18. GIASS, H.G., F. F. Snyder and E. Webster: Rate of Decline i n Resistance to Anoxia, Am. J . Physiol. li+O: 609-615, 191+U. 19. GRAHAM, B.D., Helen S. Reardon, J. L. Wilson, M. U. Tsao and . Mary L. Baumann: Physiologic and Chemical Response of Premature Infants to Oxygen-Enriched Atmosphere, Am. J . Dis. Child. 79: 371-372, 1950J Pediatrics 6: 55-71, 1950. 20. HALDANE, J.S.: Quoted by J. Barcroft: Anoxaemia, Lancet 2: . 1+85-1+89 (Sep.2+) 1920. . 21. HENDERSON, Y: Quoted by E. S. Taylor, D. Govan and W. C. Scott: Oxygen Saturation of the Blood of the Newborn as Affected by Maternal Anesthetic Agents, Am. J . Obst. & Gynec. 61: 81+0-851+, 1951. 22. HIESTAND, W.A. and Helen R. M i l l e r : Further Observations on Factors Influencing Hypoxic Resistance of Mice, Am. J. Physiol. 1^2: 310-311+, 19Ui. 23. HIESTAND, W.A., R. D. Tschirgi and Helen R. Mil l e r : The Influ- ence of Glycotropic Substances on Survival of the Primitive Respiratory Center i n the Ischaemic Rat Head, Am. J . Physiol. H+2: 153-157, 19l4l+. 29 2k° HMffiDCH, HoS.s Brain Metabolism and Berebral Disorders, Balti- i more, Williams & Wilkins, Inc., 1951 • 25o HIMWICH, H.E., Fe A* De Alexander and J e F. Fazekas* Tolerance of the Newborn to Hypoxia and Anoxia, Am. Je Physiol o 133* 327P, 19UL* 26o HIMWICH, H.E., A* 0 . Bernstein, H. Herrlich, A. Chester and J* Fe Fazekas, Mechanism for the Maintenance of Life in the Newborn during Anoxia, Am© Jo Physiol© 35* 387-391, 19li.2* ~~ 27* HIMWICH, H.E., J* F« lazefcaaeand F. A. D. Alexanders Hypoglycemia in the Intact Rat, Am. J. Physiol. 1333" 328P, 191*1* 28* HIMWICH, H,Eo, J. F. Fazekas and F. A. D« Alexanders Effects of Cyanide and Iodoacetate on Survival Period of Infant Rats, Proc. Soc. Exper. Biol. & Medo 1*6* 553-55U, 191*1. ~" r-- . . . . . . 2 9 . LeGALLGIS, J,j,C.s Experiments on the Principle of Life, and Particularly on the Principle of Motions of the Heart, and on the Seat of this Principle, Translated by N. C. and J. G. Nan ere de, Philadelphia, M* Thomas, 1813, Quoted in Reference No* (18) of this bibliography. 30© . LeLONG, M.s Foreward, Anoxia of the Newborn Infant, A Symposium Organized by the Council for international Organiza- tions of Medical Sciences, Established under the Joint Auspices of U.N.E.S.C.O. and W.H.O., xi-ocv, Oxford, Blackwell Scientific Publications, 1953. 31. LeLONG, M. and R. Laumonier* Histological and Histochemical Evolution of the Foetal Lungs Its Relation to Anoxia in Premature Infants, Anoxia of the Newborn Infant, A Symposium Organized by the Council for Internati- onal Organizations of Medical Sciences, Established under the Joint Auspices of U.N.S.S.C.O. and W.H.O., 61-68, Oxford, Blackwell Scientific Publications, 1953* 3 2 . LOMBARD, Elna A«s Electrocardiograms of Small Mammals, Am. J. Physiol. 171* 189-193, 1952;. 3 3 . MAYER, M.s Clinical Correlations in Prenatal and Postnatal Anoxia, Anoxia of the Newborn Infant, A Symposium Organized by the Council for International Organiza- tions of Medical Sciences, Established under the Joint Auspices of U.NJS.S.C.0. and W.H.O., 1 -25, Oxford, Blackwell Scientific Publications, 1953s 30 3ho McGINTY, DJUs The Regulation of Respiration, XXV Variance in Lactic Acid Metabolism in the Intact Brain, Am. J« Physiol. 88: 312-325, 1929* 35© MILLER, J»A.s Factors in Neonatal Resistance to Anoxia, I Temperature and Survival of Newborn Guinea Pigs under Anoxia, Science 110* H3-HU, 191+9* 36« MORISON, J.E.: Foetal and Neonatal Pathology, London, Butter- worth, 1952. _ 37* R20HARDS, A.G., E. Simonson and M. B. Visscher: Electrocardio- gram and Fhonocardiogram of Adult and Newborn Mice in Normal Conditions and under the Effect of Cooling, Hypoxia and Potassium, Am. J. Physiol. 17U: 293- 298, 1953* ^ 38« SCHREIBER, F«: Apnea of the Newborn and Associated Cerebral Injury, J.A.M.A. I l l : 1263-1269, 1938* 39» SEGAL, S. s unpublished data. lt.0. SELLE, W.A.: Influence of Age on Survival of Respiration, Spinal Reflexes, Pupillary Responses and ^eart Action, Proc. Soc. Exper. Biol. & Med* 1»8: Ul7-hl9, 19l|le i l l . SELLE, W.A.3 Effects of Various Chemical Agents on Survival of Primitive Respiratory Mechanism, Proc. Soc. Exper. Biol. & Medo 51: 50-52, 19l|2« U2e SELLE, tf«A«a A Simple technique for Studying the Periodicity and Survival of the Respiratory Center, Proc. Soc. Exper* Biol. & Med* 5U: 291-292, 19U3. Ii3, SELLE, WoA«: Influence of Glucose on the Gasping Pattern of Young Animals Subjected to Acute Anoxia, Am. Jo Physiol. llqs 297-300, 19hk. Ut. SELLE, WoA. and T. A. Wittens Survival of the Respiratory (Gasp- ing) Mechanism in Young Animals, Am<> J« Physiol. 133: v PlOil, 191a. — U$9 SELLE, W.A. and T. A. Mitten: Survival of Respiratory (Gasping) Mechanism Subjected to Anoxia, Proc« Soc. Exper. Biol. & Med. U7* 1495-497, 19ljl. 1|6. SMITH, CA.s The Effect of Obstetrical Anesthesia upon Oxygena- tion of Maternal and Foetal Blood, with Particular Reference to Cyclopropane, Surg. Gyneco & Obst. 69: 5811-593, 1939* ~~ 31 l+7« SMITH, CeA« and E.Kaplans Adjustment of Blood Oxygen levels In Neonatal Life, Am. J« Dis. Child. 6U: 814.3-659, 191*2. l+8o SWANN, H.G.J Studies in Resuscitation, Section m, Asphyxia Neonatorum, AF Technical Report Noa 5972, U.S. Air Force Air Materiel Command, Wright-Patterson Air Force Base, Dayton Ohio, Aug© 19l*9. 1*9© THOMS, RoH. and W. A* Hiestand: Relation of Survival Time of the Respiratory Gasping Mechanism of the Isolated Mouse Head to Age, Proco Soc© Exper© Biol* & Medo 61** 1-3, 19l*7» 50 o TYLER, D.B. and A* VanHarreveld. The Respiration of the Develop- ing Brain, Am. J. Physiol. 136 s 600-603, 191*2. 51 o WARBURG, 0 . , K» Posener and E. Kegeleins uber den Stoffwechsel der Carcinomzelle, Biochem. Ztschr. 152 : 309-31*1*, 1921*, Quoted in Reference No. (21+) of this bibliography. 520 WEGMAN, M.E.J Trend in Infant Mortality Rates, Pediatrics 6: 673-675, 1950. 53o WILSON, J . , Helen Reardon and M. Murayama. Anaerobic Metabolism in the Newborn Infant, Pediatrics Is 581-592, 191+8©


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