The ability of hypothermia to protect newborn guinea pigs from anoxic trauma

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Content THE ABILITY OF HYPOTHERMIA TO PROTECT NEWBORN GUINEA PIGS FROM ANOXIC TRAUMA by Dale Leonard Carpenter A Thesis Presented to the FACULTY OF THE GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree MASTER OF ARTS (Zoology) June 1957 UMI Number: EP67223 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. Dissertation Publishing UMI EP67223 Published by ProQuest LLC (2014). Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106- 1346 UN IV E R S ITY O F S O U T H E R N C A LIF O R N IA G R A D U A TE S C H O O L U N IV E R S IT Y PARK LO S A N G E L E S 7 This thesis, written by Bale Leonard Carpenter under the guidance of h^.f....Faculty Committee, ç ^3*^ and approved by all its members, has been pre- sented to and accepted by the Faculty of the Graduate School, in partial fulfillm ent of the requirements for the degree of Master of Arts (Zoology) Date. Dean June 19^7 Faculty Committee .. C h e à r t n a n .. e îrwu....... ACKNOVfLEDGEMENTS My most sincere appreciation is expressed to Dr. Robert Chew under whose guidance the present study was developed. Dr. Chew originally suggested the problem, and he served as a constant source of encouragement and instruction. The valuable assistance and instruction of Dr. William V. Mayer and Dr. John L. Mohr is appreciated. Thanks are also due Dr. Frederick J. Moore, The University of Southern California School of Medicine, for helpful suggestions concerning statistical analysis. Appreciation is expressed to Mr. Joseph Bamberger and Mr. Brinton Mitchell for their help and advice in the construction of the thermistor unit. My wife, Muriel Carpenter, has served in the capacity of an independent observer, animal caretaker, and in nebulous duties. Gratitude is expressed for her valuable help. TABLE OF CONTENTS CHAPTER PAGE I. INTRODUCTION.............................. 1 Statement of the Problem ................ 1 II. REVIEW OF THE LITERATURE.................. 2 Importance of the Problem .............. 2 Choice of Experimental Animal .......... 6 Availability of suitable newborn .... 6 Comparison of human and guinea pig intrauterine activities ............ 7 Anoxic survival time .................. 8 Temperature control present at birth . . 8 Previous studies ...................... 12 Hypothermia............................ 12 Hypothermia compatible with life in adult human beings.................. 12 Hypothermia in the infant............ 13 Hypothermia in other animals.......... 14- Hypothermia and Asphyxia................ 15 Cerebral Damage and Anoxia.............. 21 Asphyxia, Hypothermia, and Cerebral Damage in the Guinea Pig.............. 24- Clinical Applications of Hypothermia ... 25 Hypothermia and Asphyxia Neonatorum in the Human Being...................... 27 V CHAPTER PAGE Testing of Guinea Pigs to Determine Behavior Characteristics and Learning Abilities.......................... 30 III. MATERIALS AND METHODS OF THE EXPERIMENT . IV. RESULTS AND DISCUSSION.................. 56 V. SUMMARY AND CONCLUSIONS................ 86 LITERATURE CITED .............................. 90 APPENDIX...................................... 98 LIST OF TABLES TABLE PAGE I. Types of Temperature Control at Birth or Hatching................................ 10 II. Raw Data from Experiment.................. 99 III. t Factors and Probability, Maze Scores for Littermates............................ 60 IV. Mean Scores for Original Learning and Lowest Temperatures of All Animals that Survived to Run the Maze................ 63 V. A Comparison of the Means of Survival Times, Lowest Body Temperatures, Birth Weights and Mortality of Anoxic, Anoxic- Hypothermic and Control Guinea Pigs ... 64- LIST OF FIGURES FIGURE PAGE 1. Development of body temperature and body weight of premature infants .............. 11 2. Hypothermic survival temperatures for young warm-blooded animals .................... 16 3. The effect of incubator temperature on body temperature of premature infant .......... 29 4-. Guinea pig maternity chair................ 37 5. Sample observation data sheets for reactions of guinea p i g s .......................... 39 6. Calibration chart and diagram, temperature measuring apparatus...................... 4-2 7. Photograph of temperature measuring apparatus 4-3 8. Exposure chamber.......................... 4-5 9. Photograph of m a z e ........................ 4-8 10. Perspective drawing of maze................ 4-9 11. Maze data sheet and typical run............ 51 12. Floor plan for maze patterns............. 52 13. Comparison between number of total errors for anoxic, anoxic-hypothermic and control animals.................................. 57 14-. Lowest body temperature compared to anoxic time-run score.......................... 59 viii FIGURE PAGE 15. Birth temperatures and weights of Caesarean and normal delivery newborn .............. 65 16. Exposure time and resuscitation time .... 72 17. A comparison of phonoreception in anoxic, anoxic-hypothermic and normal (control) guinea pigs.............................. 8l CHAPTER I INTRODUCTION For many years, a difference of opinion has existed concerning the proper treatment for an anoxic newborn child. Although it has been recognized that anoxia causes cerebral damage and assumed that hypothermia might help to prevent such damage in the newborn, little work has been done to prove this hypothesis. Various workers have chosen the guinea pig as a suitable animal for study of hypothermia and asphyxia in both adult and newborn individuals, but a review of the literature has revealed only one attempt to determine whether hypothermia during asphyxia prevents cerebral damage of the type which might interfere with learning and adjustment. This study (Guerin, 194-6) was incon clusive and was never published. Guerin did not study asphyxia neonatorum. She worked with animals of various ages. Statement of the Problem The study of the effects of hypothermia on the survival and normal development of anoxic newborn animals is a vast field for experimentation. This present in vestigation is limited to the following questions: (1) Does anoxia of newborn guinea pigs cause impairment 2 of learning ability? (2) Does hypothermia during anoxia reduce or prevent this learning impairment? (3) Can any parallels be drawn between the results of this experiment and the effects of anoxia and hypothermia of newborn human beings? CHAPTER II REVIEW OF THE LITERATURE Importance of the Problem Asphyxia, which most authorities agree plays some, if not the chief role in 50 per cent of the neonatal deaths, is a major medical problem of interest to many scientific workers. If the physician is able to prevent fetal asphyxiation or to give correct treatment to the asphyxiated newborn, he may forestall later neurological problems. Schreiber (1938), Clifford (19^0), Sattler (1951) 9 Preston (194-5) 9 Blanc and Erwin (1951) 9 Courville (1955)9 and many other workers have published reports on the effects of neonatal asphyxiation. Sociologists, psychologists, and teachers are concerned with the theory that severe asphyxiation at birth may have a delayed effect on the behavior of those children who survive. Anesthetists realize that neonatal asphyxia is a special problem. Directors of maternal health, welfare bureaus, and first aid resuscitation groups are actively interested in neonatal asphyxia. The military doctor has an interest in neonatal asphyxia because knowledge of it can help him to understand the problem of warfare anoxia. The American Medical Association brought neonatal asphyxia in focus as a crucial problem. The A. M. A. 4- Committee on Asphyxiation did research on anoxia of the newborn as early as 1937, and listed asphyxia neonatorum first among asphyxiai accidents as a problem needing immediate attention. In 194-1, there was an important "round table meeting" by physicians on neonatal asphyxi ation. In 194-8, at a joint meeting of the Obstetrics Section and Gynecology Section of the A. M. A., the problem of treatment of anoxic newborn infants was dis cussed. At London, October 1-5, 1951, the Council for International Medical Sciences held a symposium on anoxia of the newborn infant (Cross, et alii. 1952). For the anesthetist, asphyxia neonatorum is of the greatest possible interest because it offers an ever present working model by which all other types of asphyxia may be approached (Flagg, 1944-). While most asphyxiai problems occur in locations and under conditions that do not offer the opportunity for study of the factors involved, asphyxia of the newborn can usually be antici pated and occurs most generally under conditions where preparations can be made to meet the emergency. The field of neonatal asphyxia could conceivably be a proving ground for anoxic therapeutics. Animal research should provide certain information which is impossible to obtain from humans. It has been recognized that there is a possible correlation between anoxia at birth and later neurological 5 manifestations indicating impairment of the intellect (Bates, 194-1; Means, 194*8; Little, et alii. 1952); how ever, there is no conclusive proof at the present time. To obtain valid data on the human it would be necessary to study identical twins: one born without asphyxia and the other suffering from asphyxia neonatorum. Only one study of a pair of twins has been reported (Preston, 194*5) • The asphyxiated twin showed neurological symptoms similar to those which Windle and Becker (194-3) and Windle, Becker, and Weil (1944) found in guinea pigs. Most studies on humans have been of the type done by Preston (194-5), i.e., starting with children with cerebral damage and working backwards to find etiological factors such as a history of asphyxia at birth. Recently, there has been a new approach to the problem as by Usdin and Weil (1952), who started with known anoxic infants and followed them into later life. They tentatively concluded that neonatal asphyxia causes delayed neurological symptoms. With this type of approach, it obviously will be many years before definite conclusions can be formu lated. Similar techniques applied to a suitable labora tory animal, such as the newborn guinea pig could possibly provide answers more quickly. 6 Choice of Experimental Animal The guinea pig lends itself to the investigation of hypothermia and anoxia of the newborn and to inferences about human newborn for several reasons. Availability of suitable newborn. A female guinea pig comes in heat and will mate within a few hours after parturition if a male is made available, therefore it is possible to know the breeding date. It is fairly easy to obtain fetuses and newborn of a desired age. The length of gestation is affected by the size of the litter; normal sized litters are usually carried about sixty-eight days, while a single fetus may be carried seventy-two days (Farris, 1950). However, single births are not frequent, and it is usually possible to obtain control animals from the same litter so that the split-litter method of study can be used, beeper (1932) and many workers since then have discussed the advantages of split-litter technique in the psychological testing of rats, and this technique has been applied to the testing of guinea pigs (Windle and Becker, 194-3). The guinea pig is a hardy animal and is easily cared for under laboratory conditions. The young can be separated from their mothers at an early age, and seem to be resistant to most respiratory diseases. 7 Comparison of human and guinea pig intrauterine activities. Placentation in the guinea pig is of dis co idal type and is otherwise similar to that of man. Certain intrauterine responses under conditions of anoxia seem to he like that of the human. Windle, et alii (1939) demonstrated, by injecting a radiopaque dye into the amniotic cavity, that intrauterine rhythmical respiratory- like movements occurred in guinea pigs under conditions of anoxemia during difficulty in labor. Guinea pig fetuses aspirated the radiopaque dye. Similar movements sometimes occur in the human fetus under anoxic states (Snyder and Rosenfeld, 1937)• Windle, Becker, and Whitehead (194-2) studied the lungs of litter mate guinea pigs that had been experi mentally asphyxiated in utero. Histological studies revealed the lungs were atelectatic, presenting compact gland-like appearance. There is a definite similarity of lung pathology in asphyxiated guinea pigs and human fetuses, as reported by Windle (1950) and Bruns and Shields (1954-). Windle, Becker, Barth, and Schulz (194-0) published a report suggesting there is a similarity between gastro intestinal activity of the guinea pig and that of the human fetus in utero. With roentgenograms they demon strated that the guinea pig fetus swallows amniotic fluid. 8 Anoxic survival time. The anoxic survival time of the guinea pig is less than that of other small experi mental animals, and appears to be close to that of the newborn human. Frazekas, Alexander, and Himwich (194-1) studied adult and newborn animals and determined that adult rats, dogs, rabbits, and guinea pigs survived after breathing undiluted nitrogen for three minutes, while the following survival times were found for other newborn animals: rats, fifty minutes; cats, twenty-five minutes; dogs, twenty-three minutes; rabbits, seventeen minutes; and guinea pigs, only seven minutes. Parmelee (1952), speaking of humans, stated, "A newborn may survive in complete apnea as long as 10-15 minutes." Parmelee also states. The newborn infant has the fortunate advantage of being able to survive a much longer period of asphyxia than an older infant owing apparently to certain anaerobic peculiarities of metabolism in the early postnatal period. Wilson, et alii (194-8) believe it is possible that infants make use of anaerobic metabolism during fetal life and for a short time after birth. Miller (1949a) considers anaerobic metabolism important in the survival of infant guinea pigs. Temperature control present at birth. The tempera ture control at birth is similar in infants and guinea pigs. The balance between heat production and heat loss 9 in the newborn human is intermediate among mammals between the well-developed guinea pig and the helpless mouse (Barbour, 194-1). However, the infant has the advantage of the superior intelligence of its species to provide a suitable environmental temperature. The newborn infant has physical control to some extent in the thermal barrier of clothing provided by the adult of the species. Pearse and Hall (1928) found that a guinea pig loses 33 per cent more heat if shorn. If the guinea pig were shorn, the physical control would be close to that of the naked infant at birth. Table I indicates the forms of tempera ture control present at birth or hatching for various animals. The guinea pig is a more suitable laboratory animal for the study of neonatal asphyxia than the immature rat or mouse which have poor temperature control at birth. There is evidence that newborn infants, if left to do so, will establish a body temperature in equilibrium with their environment, but at a lower than the "normal" rectal temperature of 99° to 100° F. (37.2° to 37.8° C.). Only after several weeks is relative equilibrium at the 99° to 100° F. level established (Figure 1, page 11), and some premature infants are even slower in establishing this temperature level. 10 TABLE I TYPES OF TEMPERATURE CONTROL AT BIRTH OR HATCHING (From Yapp, 1939) Animal Chemical control Physical control Human + - Guinea pig + Chick + + Mouse - - Pigeon - - Rabbit + - Note the lack of physical control in the newborn infant. 11 ^ ec^ al (Jrtzuhaior Tc^nperaTure =-%'*g) 3Llf96 3&1-97 35.6' 96 3# 95 344 33.% 333 [MtqSUwnei\^ ^46 u;fW> ÉL FâK*'e*^^®4 4hep»n6fnw€r. Wo+e +ha+ +he44\epmonr>e+cr Cowl A not re^Gfeh bejow 9l®^ ^Ifhcoqh, fheiempera^ore oç -the fin n i matj have oeen beiovs??/^ — ---- 1 ----— —' ---- - A t C From Gordon^ (9&U 50 60, c* 'Sl.Z' Ç O ■99 5 8odt* weight ( N)o tV>cob<cTot' vMas osed)_ — 0 Pôdt^TempewTürejrectal ^ sty •98 36| ■37 35L y 354 •95 ...... ' : 33^- •9r —a 5 _ l -----6 2 _ | _____L S j_; _ ao» S£i 3Û, ^.5 -.4 -5 01- C Fwiv\ f7e»rdo#\^ and (orahain^ ^^0 Figure 1. Development of body temperature and body weight in premature infants. 12 Figure lA, indicates the need for a clinical thermometer reading as low as 77° F. (25° C.). Gordon states, in speaking of this temperature chart, that the course of this infant's temperature was "below 92° F. (33*3° C.), the lowest reading on our clinical thermometer for almost three weeks." Just how low the temperature was for these critical first three weeks will never be known. Gordon also says, "Many of our small infants have temperature curves like this one." Previous studies. Previous studies of hypothermia and neonatal asphyxiation have been done with guinea pigs. The use of the guinea pig in the present study allows comparison of results with those of previous investiga tions . Although it is impossible to select an animal which would react to neonatal asphyxia in a manner comparable to that of the infant in all respects, the previous review of similar characteristics indicates that the guinea pig is suitable for this study. It is appreciated, however, that there are many difficulties in formulating theories for human reactions based on animal data. Hypothermia Hypothermia compatible with life in adult human beings ranges between 96.8° F. (36° C.) and 75.2° F. 13 (24° C.). The lower limit is based on the data from uncivilized Dachau experiments during World War II (Alexander, 1945). A body temperature of 75.2° F, (24° C.), or even less, can be survived if hypothermia is very carefully controlled, since Bigelow et alii (1954) performed operations on humans at temperatures of 68° to 87.8° F. (20° to 31° C.). However, Ciocatto and Cattaneo (1956) state that temperatures below 77° F. (25° C.) are unsafe. Hypothermia in the infant is difficult to define since the "normal temperature" for an infant is a debatable value. Miller (1949a) states, "All newborn animals (including the human) are poikilothermic with body temperature varying with ambient temperature." If this is true, then the human newborn might have a lower survival temperature than 75.2° F. (24° C.). Various workers have stated that babies can withstand extremely low temperatures. Crosse (1955) stated that some babies at the Sorrento Maternity Hospital in Birmingham, England, have survived after rectal temperatures of 82° F. (27.8° C.) Eckstein (I926) reported temperatures as low as 78.8° F. (26° C.) in newborn humans. Newspapers often report rectal temperatures as low as 68° F. (20° C.) in newborn infants with complete revival. Klein (1936) reported the recovery of a one-month old infant from 14 64.4° F. (18° C.) rectal temperature. Hicks ^ alii (1934) study found that Australian aborigines could sleep naked on the ground, protected by a small windbreak and a small fire, in temperatures of 35.6° to 50° F. (2° to 10° C.) without shivering and with out increasing their metabolic rate. This has been attributed partly to lack of autonomic control. It is known that premature infants have a similar lack of autonomic control (Parmalee, 1954). The present writer has observed premature infants with such poor autonomic control of their superficial blood vessels that, after lying on one side for an hour, the lower side would be hyperemic while the upper side would be blanched. There was a sharp line of demarcation between the two halves of the body. Hypothermia in other animals has been a subject of interest for many years. Many references, for example, Boyle (1665), were made to heat and cold as it affected animals long before the eighteenth century when Thompson tried to make quantitative measurements of heat. The recent literature on hibernation and low temperatures in animals is voluminous. As early as 1824 Edwards was concerned with hypothermia and temperature control in newborn animals. He studied many animals and found that the guinea pig had 15 fairly well-developed temperature control at birth. Pembry (1895) studied newborn guinea pigs and their ability to maintain body temperature in relationship to their output of carbonic acid. Figure 2 illustrates the ability of young warm blooded animals to control body temperature at low envirorb- mental temperatures. Survival temperatures indicate that the ability to control body temperature under hypothermic conditions is variable and is acquired rather suddenly in such species as the rat and hamster, while the transition is rather gradual in the guinea pig. Some lower levels of survival for the human are also presented in Figure 2. Hypothermia and Asphyxia Hypothermia and asphyxia in the guinea pig have been subjects of investigation by various workers in recent years. Their publications will be discussed in detail because they furnish a portion of the background and stimulus for this thesis. Of all the investigators, James Miller, Jr. and his co-workers seem to be the most active. Miller reported (1949a) that he had reduced the body temperature of guinea pigs by exposing them to air currents from a twelve inch fan after wetting all but the head and anal regions with 95 per cent alcohol. When the colonic temperature had fallen 2° to 5° C., the guinea pig 16 ÿ I s. £ c ~o ■ o 09 o 0 F c St- ■30- 7n- 25- 68. Z0- 59 ■»5- 30 ■ 10- 41 ■ 5 - Uajaa&ter 35’A c k » * + - Sü'f'vÿva I "ifitmperalures 4or Vû < * v > ^ - blooded Qm'mals (.Prom Adolpb^ 195/) Survival lempera-lures^ ko wan % 0 ( Fnom Onosse^ 195"$) A (Frovn ( S > CFrow^ Kleih^/9S9 0 ( Fro m /V(cyander Figure 2. Hypothermic survival temperatures for young warm-blooded animals. 17 was placed under a bell jar and exposed to an atmosphere of 95 per cent nitrogen and 5 per cent carbon dioxide. He kept a record of visible reactions with a stop watch and recorded the time of death as compared to littermates exposed to nitrogen asphyxiation without being cooled. He concluded that a reduction in temperature prolonged survival of asphyxiated day-old guinea pigs. After these preliminary experiments, Miller (1949b) tested more newborn guinea pigs in a similar way. He noted a 185 per cent increase in mean survival time for animals cooled to 51.9° F. (11° C.) before asphyxiation. This represented an increase in survival time of 7.1 per cent for each degree cooled below normal, 99.5° F. 37.5° c.). In other experiments. Miller (1949b) found that, at comparable temperatures, young adult guinea pigs lived one half as long as newborn. He concluded that even adult guinea pigs behave like poikilothermal animals when subjected to severe anoxia, and suggested that a reduc tion in body temperature during anoxic states might prolong life. To prevent stimulation of motor activity. Miller tried giving nembutal before hypothermia. He concluded that this was effective in increasing anoxic survival time in some cases. Miller, Miller, and Farrar (1949) reported an experiment in which they injected 3 per cent hydrogen 18 peroxide intraperltoneally. They concluded that this treatment prolonged survival time and speeded recovery in anoxic day-old guinea pigs. They determined recovery by timing stages of recovery which were: (1) reappearance of breathing, (2) attempt and completion of the righting reflex, and (3) crawling. They believed that the most reliable sign of recovery was crawling activity. Miller, Miller, and Farrar (1950) reported that during asphyxiation lactic acid accumulates and adenosine- triphosphate resynthesis is impaired. They injected ATP into anoxic guinea pigs and concluded that ATP injections might be beneficial in the treatment of asphyxia neonatorum. Miller and Miller (1953) determined that the beneficial effects of nembutal narcosis in increasing resistance to asphyxia is augmented as hypothermia increases; and, they concluded that hypothermia will prove effective in treating asphyxia neonatorum. Miller, Miller, and Farrar (1954) summarized their findings in previous experiments and added additional data to confirm their previous conclusions. They reported that 385 neonatal guinea pigs were tested for the effects of hypothermia and hyperthermia on asphyxia in 95 per cent nitrogen plus 5 per cent carbon dioxide. Body tempera tures above 110.3° F. (43.5° 0.) caused death before asphyxia, and some animals with temperatures below 68° F. 19 (20° C.) also died before asphyxia. The survival of animals below 68° F. (20° 0.) was most variable. When cooling was started during asphyxiation, the temperature drop was approximately 50° F. (10° 0.) greater in the experimental animals than in the controls. Rapid warming (in a bath at 113° F. (45° C.) of cooled, severely asphyxiated animals caused a more rapid recovery of those animals which survived, but did not increase the total number of survivors. Autopsy revealed congestion in the lungs, subcapsular hemorrhage in the adrenals, hemorrhages of the testes or uteri, and occasional hemorrhage of the digestive tract and coronary arteries. Although Miller and his associates have made considerable investigations of hypothermia and neonatal asphyxiation, they have not studied differences in overt behavior between guinea pigs in control and experimental groups. Gosselin (1949) investigated hypothermia in adult guinea pigs to determine whether the diminution of oxygen consumption during hypothermia is relatively greater than the concomitant reduction in oxygen requirement. Gosselin subjected guinea pigs to hypothermia by placing them in rubber jackets and immersing them in ice water. In the early stages of cooling, it was found that there was increased struggling, hyperpnea, enhanced metabolism, and slowing of heart rate. When cooling was produced at 20 0.2^ to 0.3^ C. per minute, maximum ventilation occurred at 91.4° to 98.6° F. (33° to 37° C.) (colonic temperature) and was 170 per cent of the precooling rate. Oxygen con sumption was also I70 per cent of the precooling rate. Below 91.4° F. (33° C.), however, both of these factors diminished. In proportion to the oxygen consumption rate, the ventilation rate remained high until terminal apnea; so it was concluded the progressive fall in oxygen con sumption under hypothermia was not due to failure of breathing. Gosselin believed that unless the chilled animal was properly rewarmed, damage sustained during severe chilling would impair thermal adjustment of the guinea pig at a later date. However, she did not make any specific recommendations as to technique for rewarming. Bigelow et alii (1954) used water baths, hot water bottles and diathermy to rewarm those patients cooled be low 82.4° F. (28° C.), and suggested rapid rewarming until 91.4° F. (33° Cj was reached. Penrod and Rosenhain (1951) state that it has been well established that functions of homiothermic animals show an almost linear decline with decreasing body tempera ture. Rosomoff and Holaday (1954) state that cerebral oxygen consumption and blood flow vary proportionately with body temperature during hypothermia. Because the 21 arterio-venous oxygen difference appears to be unchanged during cooling, they conclude that hypothermia probably does not produce hypoxia of the brain tissue as long as adequate respiratory and cardiac activity is maintained. Bigelow, Mustard, and Evans (195^) stated that oxygen consumption in human beings is reduced by half at a temperature of 86° F. (30° C.) during anesthesia. Apparently, from the literature just discussed, it is possible to postulate that hypothermia does not cause failure of breathing, hypoxia, or an increase in cerebral oxygen requirements after the initial stage of cooling. Cerebral Damage and Anoxia Yant et alii (1934) found that anoxemia in dogs caused severe hyperemia and circulatory stasis in the brain stem. Cells found to be most sensitive to anoxemia were the outer cells of the cortex, cells of the thalamus, the visceral efferent nuclei, and the sensory and association neurons of the brain stem. Thorner and Lewy (1940) exposed cats and guinea pigs to sublethal periods of anoxia in a nitrogen atmosphere and found that the guinea pigs behaved in a stereotyped manner. During the first ten to fifteen seconds they became hyperactive, and in another twenty seconds the animals showed the following symptoms ; arching of neck, slumping to the floor, loss of perception 22 of sound, loss of light reflex, cyanosis of mucous membranes, and spasmodic movements varying from clonic twitchings to generalized convulsions. After one minute, the guinea pigs were apneic and needed artificial respira tion, but in no case did the heart cease beating until after respiratory failure. Lethal exposure time was 105 seconds. Brains of the cats and guinea pigs were examined at various intervals following revival from asphyxia, and evidence of cerebral damage was found in fibrosis of the meninges and blood vessels. Perhaps the most interesting observation was that the cats and guinea pigs showed no evidence of functional impairment although severe brain lesions were found. In man, also, severe lesions caused by repeated anoxia can exist without the individual show ing functional disturbances. Thorner and Lewy attributed the lack of detectable neurological symptoms in lower animals and man partly to present insensitive diagnostic methods. Bunch (1952) exposed white rats to 3 per cent oxy gen for thirty minutes after birth and compared them with white rats that had been subjected to anoxia by exposing their mothers to oxygen lack for two hours before their birth. Litter-mate controls in each series were used. After approximately seventy days, the animals were started on learning experiments in a multiple T-l4 unit water maze. Retention was tested after an interval of 23 thirty days. He concluded that the ability of white rats to learn and remember a difficult maze problem is not affected by severe anoxia for thirty minutes within two hours after birth. However, the effects of severe anoxia during the fetal period greatly affected the learning ability and retention. Hurder and Sanders (1953) studied the effects of anoxia on rats starting the first hour after birth. Split- litter controls were used. A Stone-Multiple T maze was used. They concluded that rats in infancy exposed to simulated altitudes of 30,000 feet for three hours (pOg 4? mm. Hg.) were less affected functionally than adult rats. Heymans (1950) reported studies on cats, rabbits, mice, and guinea pigs. He found that various portions of the nervous system were able to withstand anoxic anemia for periods of time varying with the age of the animal, the state of activity, and the level of blood sugar. Vitamins, thiourea, thyroxin, adrenal cortical substances, sodium pentobarbital, and certain diseases also affected the survival of nervous tissue in anoxia. He found that synaptic function showed remarkable resistance to anoxia, and that the pe ripheral nervous system remained excitable in anoxia longer than other nervous tissues. Many other studies of anoxia can be found in the literature, but the studies discussed above seemed 24 especially pertinent to this investigation. Asphyxia. Hypothermia, and Cerebral Damage in the Guinea Pig. Windle and Becker (1943) used a simple alternation type maze with food and cover as motivating factors to test guinea pigs that had undergone experimental asphyxia tion at birth. They found definite differences between the behavior and learning ability of the control animals and the asphyxiated animals. The control animals were able to learn quickly that the exit was always behind a blind alley in the maze, while the asphyxiated animals learned the exit more slowly. Windle, Becker, and Weil (1944) compared the brains of experimentally asphyxiated animals and their litter- mate controls and observed definite pathological changes in the brains of those asphyxiated animals that either failed to learn the solution to a simple maze or quickly forgot the solution once learned. Often there was extensive nerve-cell loss in sensory areas such as the thalamic nuclei and geniculate bodies, while in other cases the damage was predominately cortical. Windle, Becker, and co-workers did such commendable work with asphyxia neonatorum that it was a great loss to this field when it became necessary for them to interrupt their studies and direct their research along other lines 25 during the war. However, Windle (1955) states that, at the present time, he is organizing a project in his laboratory at the National Institute of Neurological Diseases and Blindness. He expects to extend the work that Becker and he did and to try to relate it to cerebral palsy in man. Guerin (1946) exposed hypothermic and "normal" temperature guinea pigs to asphyxiation in nitrogen atmosphere, and then compared control and experimental animals according to their behavior in a maze. She used a modification of the maze used by Windle and Becker (1943). Guerin concluded that chilled-asphyxiated guinea pigs made fewer errors and ran the maze in a shorter time than did "normal" temperature asphyxiated guinea pigs. She also concluded that chilled guinea pigs could stand asphyxiation longer than guinea pigs asphyxiated at "normal" temperatures, but did not define "normal" temperature. Guerin used guinea pigs of varying age, apparently from two weeks to maturity. Because of this fact, it would be difficult to compare the results of her experiment with an investigation involving the use of newborn guinea pigs. Clinical Applications of Hypothermia In the human, hypothermia due to pathological causes does not occur as often as hyperthermia. 26 consequently little information is available concerning the causes of hypothermia. Recently, however, artificial cooling has been used in the treatment of cancer and during surgery on the heart. The prolonged cooling apparently does not cause unsatisfactory physiologic results. In France, artificial hibernation has been used to tide children over various crises in childhood diseases. Lacomme (1954) has used hypothermia to decrease oxygen demand and to avoid prolonged anoxia in neonatal condi tions, such as erythroblastosis, toxicosis, trauma of birth, transfusion reactions, and prematurity. He administered chlorpromazine subcutaneously every forty minutes until the infant ceased to regulate body tempera ture. Then the infant’s body temperature was regulated in an incubator set at 8o.6° to 82.4° F. (27° to 28° C.). Restoration of body temperature was begun not later than the third day with smaller and less frequent injections. The author claimed great success with this technique of hypothermia. Crosse (1955) stated that she had used this technique to lower metabolic rate in premature anoxic infants, but had not yet used it in sufficient cases to determine its value. 27 Hypothermia and Asphyxia Neonatorum in the Human Being A review of current literature indicates that fetal and neonatal asphyxia have been studied by various research groups. Few investigators have studied the effect of hypothermia on cerebral damage. Although there have been a few empiric clinical applications (Lacomme, 1954), there is no conclusive proof that hypothermia actually prevents cerebral damage. Segal (1954) stated that there seems to be a lack of evidence bearing directly on a relationship between hypothermia and prevention of asphyxiai damage at birth. Various workers disagree with the belief that hypothermia would be valuable in neonatal asphyxia. Smith (1954) thought severe hypothermia would be of little advantage and would probably be damaging to the insuf ficiently expanded lungs of the human newborn. At the symposium on neonatal asphyxia, conducted by the Council for International Medical Sciences, 1951, the general opinion was that hypothermia would not be beneficial to anoxic newborn infants (Cross et alii, 1952). The empirical and traditional routine, accepted for centuries by midwives and obstetricians, has been to place the newborn child in a warm environment. Not only full term babies, but also cyanotic and premature infants have been treated in this customary way. Although practical consequences preclude any rash departures from 28 this empirical approach, there are reasons why warming of the anoxic newborn infant seems to be an illogical ap proach to treatment. Placing an asphyxiated or even mildly anoxic new born in a warm bassinet, incubator, or pressure chamber, and giving it strong concentrations of oxygen does not seem consistent with certain accepted physiological facts. Warming produces an increased need for oxygen (Eckstein, 1926) . The high concentration of oxygen may in itself be damaging. Recently it has been demonstrated that high concentrations of oxygen injure guinea pigs’ lungs and promote the formation of hyaline membranes similar to lesions seen in the lungs of premature infants (Bruns and Shields, 1954). High concentrations of oxygen, especially with premature infants, have been found to cause blindness and retrolental fibroplasia (Crosse and Evans, 1952). Figure 3, taken from Reardon, et alii (1951) shows that a high incubator temperature must be maintained to sustain an infant’s body temperature at the "normal level." It would appear that sometimes an artificial temperature environment is imposed on the poikilothermic human newborn. In view of the accumulated evidence that as the body temperature rises the oxygen consumption becomes more and that the newborn is poikilothermic, it appears that the practice of warming anoxic newborn 29 "F %8 l o e . M' (00- j&D- 36*0- 84 {Z6.D- 74- ;io.o ' “®"^^^rema4ure>Xia-fiirrffe l3oiu 'T^mjoe^a-hjpe. 0 -0 - 'TnCo k tfor ^T e in |je n t4 o p e * W V \ r ^ f? ^3 4 Octets Pd6+na+a| 5 6 " 7 8 3 /on /i »3 <4 » 5 ' (From Reardon Wrisen and G rahamJ9S^ Note that this figure seems to indicate that a high environmental temperature has been imposed on the premature infant during the first six days of life. This chart shows that by trying to maintain a "normal temperature" for the infant, the incubator temperature had to be kept at a high level. Figure 3. The effect of incubator temperature on the body temperature of premature infant. (From Reardon, et alii. 1951) 30 infants is a problem for conjecture. Cooling, rather than warming would be a more logical therapy for the anoxic, poikilothermic newborn. Testing of Guinea Pigs to Determine Behavior Character istics and Learning Abilities Although the guinea pig is a most appropriate animal for investigating the effects of hypothermia on anoxic newborn, it is a difficult animal to use to determine cerebral damage. Relatively few investigators have been interested in determining the intellectual power of the guinea pig. So, there are very few standard techniques for testing learning, memory, or neurological damage in the guinea pig, and the guinea pig is often not adaptable to techniques established for mice and rats. Becker (1946) gives an excellent review of information on the behavior of species of Cavia. Allen (1904) studied guinea pigs* reactions in problem boxes. She demonstrated fairly conclusively that memory exists in the guinea pig at three days of age. Hadley (1927) made discrimination studies in the guinea pig and showed that the adult guinea pig can be taught to distinguish between stimuli of similar form, but of dif ferent sizes. Muenzinger (1928) found that guinea pigs are superior to rats in problem boxes where burrowing under a barrier is involved. Muenzinger (1928) and 31 Becker (1946) have found that guinea pigs are unable to solve at the same problem level as the rat, but are more adept in finding several solutions to simple problems. Almost every investigator of the guinea pig believes that it is less stereotyped in behavior than the rat. Even though the guinea pig is superior in some respects to the rat in maze experiments, it has many limitations. Conditioning experiments are difficult be cause it takes a long time to condition a guinea pig, and few investigators have used this technique (Grindley, 1932; Horton, 1933). The habits of guinea pigs limit the type of maze that can be used and the motivation for running the maze. In its natural habitat, the guinea pig is a grazing animal and hides in tall grass or other suitable cover when danger approaches; therefore, suitable cover should be an inducement for the guinea pig to run a maze. A guinea pig will seek water when deprived of it for a short time if it has been fed on dry food. In a hot environment, it will bathe as it drinks and will place a part of its body in the water when not thirsty. The present worker observed that two week old guinea pigs, after they had apparently satisfied their thirst, would again drink heartily if placed in a brightly lighted box. The guinea pig is a constant feeder as compared to the rat which normally feeds once a day (Becker, 1946). 32 However, the guinea pig will not often seek food when placed in a strange environment even if it has been starved, while the rat or mouse can be starved and will perform well in a maze to obtain a reward of food. The guinea pig chooses carrots when offered a variety of foods, freshly cut moist carrots attract it more readily than dry carrots. Becker (1946) stated that sex is a strong drive in the guinea pig and that a buck will cross a high voltage barrier to reach a female in heat, although the same buck will balk at crossing a much lower voltage barrier to reach food. The rat does not seem to be disturbed by extraneous noises as much as the guinea pig, which has a tendency to huddle against the side of a maze and to listen for strange noises. The guinea pig does not seem to be as inquisitive as the rat or mouse. Guinea pigs are not climbing animals and also are not as adept as rats at opening doors or pulling weights. They are not as adaptable to an elevated maze. Becker (1946) used a rectangular box as a maze. A covered starting box was provided at one end and two covered goal pens at the other end. Two right-angle blinds were arranged so that they could be set up at random or in such a way that the guinea pig would have to run the maze, right-left, or left-right to reach the 33 alfalfa filled goal pens. A T-blind was also placed in front of the two goal pens, which were built with sliding glass doors. Electric grids were placed in the starting box and on the floor of each blind alley. Indirect ob servation of the guinea pig was made in an overhead mirror. The guinea pig was first taught to run the maze without the two right-angle blinds; then the blinds were placed into the box, and the animal was trained to circumvent the three blind alleys in order to reach the attractive goal pens. It was taught that the entrance to the goal pen was always behind the last blind alley. Becker kept total trial and error scores for comparative data. Guerin (1946) used a similar maze but with only one right-angle blind. CHAPTER III MATERIALS AND METHODS OF THE EXPERIMENT Introduction The experimental procedure of this study consisted of two main phases: (1) exposure of newborn guinea pigs to conditions of anoxia or anoxia and hypothermia, and (2) the testing of these guinea pigs for any abnormalities of behavior which developed following anoxia or anoxia- hypothermia. Normally delivered and Caesarean section guinea pigs were studied. One littermate of each group was exposed to anoxia, and one of its siblings was exposed to anoxia in conjunction with cooling. Whenever the litters were large enough, one sibling was saved for a control animal and was not exposed to either hypothermia or anoxia. A total of 67 newborn guinea pigs was used. Of the 67, 23 were delivered by Caesarean section and 44 were born by normal delivery. In the Caesarean section group, 11 were exposed to anoxia, 9 were exposed to anoxia and hypothermia, and 3 were used as controls. Normally delivered guinea pigs were divided into groups as follows: anoxia, l4; anoxia and hypothermia, l4; and control, 16 animals. 35 Both anoxic and anoxic-hypothermic groups of ani mals were exposed to asphyxia in a nitrogen atmosphere until detectable heartbeats and breathing movements ceased. The animals were revived by gently and intermit tently blowing into the animals' mouths via a glass T tube. The side arm of this T was left open to prevent overinflation of the animals* lungs. Auditory, maze, and other neurological tests were used to determine the effects of the experimental con ditions on the animals. Tissues were saved from a few representative animals and examined. It is planned to make a further study of these tissues in a future inves tigation. Caesarean Section Guinea Pies Guinea pigs delivered by Caesarean sections were included in this study to obviate the possibility that birth injury and anoxemia during labor might affect the results of the experiment. To obtain Caesarean section animals, sixty-eight to seventy-two day old guinea pig fetuses were delivered by the same general procedure as that employed in human Caesarean sections performed under local anesthesia. Obviously a few modifications in technique were necessary. A type of maternity chair was used as a support for the guinea pig during the Caesarean section, since it 36 was observed that mother guinea pigs delivered their young in a sitting position. All other positions tried in the preliminary Caesarean sections caused struggling. The maternity chair (Figure 4) was covered with a cloth, and the animal was taped to the side supports with masking tape. The mother * s abdomen was shaved and cleaned one- half hour before the operation. The abdominal skin and muscles were anesthetized with 1 per cent procain with adrenalin 5 to 10 minims to every 150 cubic centimeters, Procain was injected into the skin, fascia, and muscles as the Caesarean section progressed. The uterus was exposed by a midline incision and all fetuses were delivered as quickly as possible. As far as it was pos sible to determine, all fetuses used in the experiment were delivered quickly enough to prevent aspiration of amniotic fluid. As soon as the newborn had the membranes removed from their faces, most of them began spontaneous breath ing. Those that were slow to respond were given artificial respiration by the mouth and T tube technique. Immediately after the newborn had been wiped dry and the cords had been clamped, the mother was sacrificed with chloroform. During the delivery, the mother guinea pig did not squeal or struggle. 37 Figure 4. Guinea pig maternity chair. 38 The motherless Caesarean animals were fed a mixture of one half cow*s milk and one half water. Most of them readily learned to suck from an eyedropper. Some started eating solid Purina rabbit pellets within the first few hours, while others had to be placed with foster mothers. It was found that many of the less active Caesarean section guinea pigs had to be stimulated in order to start breathing. Rubbing their genital areas and twisting their ears was found to be an effective stimulus. All of the Caesarean section guinea pigs had a better chance for survival when stimulated just prior to feeding. Normal delivery newborn were obtained as a rule after a sixty-eight to seventy-two day gestation period. When possible, litters were selected which had three or more normal animals. This allowed use of a split-litter technique. General Observations Reactions of the guinea pigs from the time of natural or Caesarean birth were recorded on mimeographed check sheets. Figure 5 is a facsimile of this check sheet. This check sheet was based on the excellent work of Avery (1928) concerning responses of newborn guinea pigs and also that of Allen (1904) and Becker (1946). 29 Anim al N L .------ ^ or $ AnoXlec — *• Wdfltftb^rrhiC----- Ckesoreon ----- Niorm ol (bliuenx ------ Pu)S€. p *r m'miAc. Tbfnpevti+ure^ (L . * < * 1 r. G>tamS M Çft<*+b<a+ p«ur, . mmu+e. j O ifim 'pîW yisk R « & p ira ^ « ^ per Jtiinu+e*.. 3 “Jum p IcK '' T o necis »4i«|h Pi4ck, Moves & r c C o w Pi Veh , T urns K«iiM . c . o > Fcfes open^ fJonnel or A bnimiel CI& reFlejc -fo Touch Pupaiaru W Itft UalrtV* ■ Î ur « £ 5 (4eXt, 4^0-41^^" C . %*4,Con4#&ekwvi CcH’ . s - r t 'c . ■T^+- <!orrtmcVien aJfaetoryj +o onise .a 1 V . O . o C Leof 4inK4ed Fore l*q&- First Xmxied ia4e O r^m W wi fla tte r Si^e 4o f«4-. PNCK e«u»«s <?H A +rae4i6rk *4 Pkc+ri’ «nl +o-(W , _ V^caiiza^iftii ^SCM+ek r<Pfcc,noce {kap at- cu ia 4 c ir on n â S t . 'i môf^er ci- 1 V } K 0 • i 1 V . « O 8ucK& CKciret4eri s4-& c, 7 Avoids Qflp4urc (V je»+e of fÉtteito/Ü N ote «.f o+Aff rfo*4'*0S M h < of 0+h*r v/PAt+ioiS Figure 5. Sample observation data sheets for reactions of guinea Plg3. 4o The responses listed on the check sheet are those of normal newborn guinea pigs. No notations were made on the check sheet except pulse, respirations, and tempera tures , if the animals were normal at birth. Abnormal new born animals were discarded. The tabulation sheet served as a check on the normality immediately after birth, as well as providing a check list for abnormalities which might develop during the remainder of the experiment. These tests seemed to be adequate for this experiment, but could be vastly improved. Because some of the animals indicated phonorecep- tion loss that could not be adequately tested with tuning forks, they were tested with an improvised tape recorder audiometer. The experimental procedure and equipment used in the tape recorder audiometric testing was as follows: Various frequencies from 60 c.p.s. to 10,000 c.p.s. were recorded. A Heath-Kit model A-01 audio oscillator and Webcor Model 2130 tape recorder were used. The guinea pig's response to these frequencies was tested in a box with a sponge rubber floor covering. This box did not restrain the animal, and the animal was free to leave the box at any time during the test that it decided to do so. This box with the animal in it was placed next to the tape recorder. Each frequency was sounded intermittently, and the volume was steadily increased until the guinea pig first detectably moved its ears. Normal guinea pigs* 4l responses were taken as a standard for comparing the traumatized guinea pigs. Anoxia with Hypothermia Procedures Within the first day after birth, each guinea pig that was to be used for the anoxia with hypothermia experiment was made hypothermic by wetting all but its face and perianal region with 95 per cent ethyl alcohol, and exposing it to direct air currents from an electric fan (Technique of Miller, 1949a). The animal was placed in the nitrogen exposure chamber while still partly wet, and asphyxia was begun while the last part of the cooling was occurring. This procedure prevented shivering in most instances. During cooling, during exposure to anoxia, and during recovery, the animal's colonic temperature was measured with a small thermistor. Temperatures were read directly from a calibrated microammeter connected to a battery-operated bridge amplifier with two CK 72 transis tors. The instrument was calibrated frequently against a standard Fahrenheit-centigrade glass thermometer to check its stability. Although the calibration curve varied some, it was found to be essentially linear. Figure 6 presents a sample calibration chart and diagram of the instrument, and Figure 7> page 43, is a photograph of the apparatus. ^2 CK arAC+ens-kic 0«libw4ion Curve, e Gal*ibra4ioir» fs t. i% . X C<t il brû4ion Figure 6. Calibration chart and diagram, temperature measuring apparatus. TfcermisW A Jt/ # $ 0 =JËL _o_ C O to 00 Figure 7* Photograph of temperature measuring apparatus. 44 The thermistor was placed approximately 12 milli meters into the colon. During preliminary experiments, it had been determined that this was the area which most accurately recorded the temperature of the lower colon. When the animal*s temperature had fallen to the range of 89.6° to 73.4° F. (32° to 23° C.), the animal was placed in a small chamber (Figure 8), and exposed to a continuous stream of filtered nitrogen. The nitrogen flowed indirectly into the chamber so that it did not blow on the animal, â bleeding mechanism (the sponge rubber gasket on the exposure chamber door) was provided so that the pressure in the chamber approximated atmospheric pressure. The animal was supported on a piece of sponge rubber in which a crystal microphone was embedded, â piece of sponge rubber was also placed over the animal, and the two pieces of sponge rubber taped together with masking tape. This arrangement allowed free breathing movements, yet held the animal securely. The thermistor lead wire was held in place with a piece of masking tape. Heart and respiratory sounds were recorded directly on tape via the tape recorder microphone in the exposure chamber. Visual observations of body movements, other pertinent data, and time signals were recorded on the same tape by the use of a lapel microphone and a Switchcraft "Y" mixer. 45 Figure 8. Exposure chamber 46 Recordings were transcribed at a later time. To facilitate the detection of heart beats, recordings were made at inches per second and played back at 3 3/^ inches per second. When voice data were transcribed, the tape was played at the original recording speed of 7i inches per second. After beating of the heart was no longer audible, the hypothermic animal was further exposed to anoxia until breathing movements were no longer visible. A character istic last gasp was the criterion for removal from the asphyxiation chamber. This last gasp can best be described as an explosive inspiration accompanied by hyperextension of the spine and very obvious contractions of the facial muscles. This last gasp usually occurred after a period of apnea, which had been preceded by a period of quiet, slow shallow breathing. Animals left in the nitrogen beyond this last gasp could be revived but then died within ten days. After the last gasp, the animal was removed from the chamber and revived with the artificial respiration technique previously described. Hypothermic guinea pigs were rewarmed (at approximately 104^ F. (4o^ C.)) with radiant heat from an electric heat lamp. When its temperature reached normal, the animal was returned to the cage with its siblings. The animals were observed and tested for normal reactions at frequent intervals after 4? anoxia. Anoxic Procedures The procedure for the anoxic animals was exactly the same as the anoxic-hypothermic procedure, except the anoxic animals were not made hypothermic. However, some animals showed a decrease in body temperature during anoxia even though they were not artifically cooled. Control Procedure Control animals were observed and tested for normal reactions but were not exposed to either anoxia or hypo thermia. Maze Testing After the animals had attained a weight of 375 to 600 grams, they were trained to run a maze. Similar weight was taken as an indication of probable equal development. The maze. Figures 9 and 10, is similar to that used by Becker (1946). The inside of the maze was painted white, while the inside of the goal box was painted grass green. All of the floor area of the maze was provided with strips of brass. Alternate strips were inter connected, and the entire grids were connected to a dry cell battery, simple key, and inductorium. If the animal were to step on any two brass strips, it could be shocked 48 Figure 9. Photograph of maze 49 Figure 10. Perspective drawing of maze. 50 with a faradic shock if the experimenter so desired. The gates were operated at a distance with guide lines. The maze was uniformly lighted. Time for each run was measured in seconds. Seclusion, cover, freshly cut carrots, and safety from electric shock were used as motivating factors to get the guinea pig to run the maze. All actions of the animal were observed in the overhead mirror. The progress of the animal through the maze was charted on mimeographed floor plans of the maze. Figure 11 shows the floor plans and a typical run that has been recorded. A different color pencil was used to record each trial. This method allowed the maze pathways to be studied in detail at a later time. The following maze patterns were used: open"run, right-left run, left-right run, random alternate sequence run, and a retention random alternate sequence run. Figure 12, page 52, illustrates the floor plan for each of these runs. Open Run. The guinea pig was placed in the covered starting box and gate ”A*‘ opened. If necessary, an electric shock was given to insure departure of the guinea pig from this starting box. The animal was taught to run the maze without the two rightwangle blinds (1 and 2) and with both glass doors open to the goal boxes (4^ and 42), either one of which was considered a correct goal. 51 Animal Wo.— i , ( i o n - V l i f t i — %c — : 0 4 l ‘ Remarks_____________ Ur.m&tfji Ai .0 ÙZzL Ron CRen\arW:s -for ail runs^ Figure 11. Maze data sheet and typical run. 52 Lef+ ^oy 4% GUSS boor pal ► y □ l / / G a W fil TT ^arcrr — ^ rhe Star+incj-l3o>c ^ Ôtt-1-e \was ope^ej To Uef -the aomacL P/q Ou4, lhen IU<^5 Closed/ Sfarliw 8 < y ( GldSS 6 c f l | ^6<>X Doop C.losod Right-Left Run Pattern Left-Right Run Pattern Figure 12. Floor plan for maze patterns. These two patterns, i.e., R-L and L-R, were alternated in random order for the Alternate-Sequence Run and the Retention Test. The Open Run was a pattern without blinds 1 and 2 in the runway, and with both goal box gates open. 53 The learning process was continued until the animal met a criterion run of no errors, one goal box attempt (i.e., no goal box errors), one shock, and ten seconds running time. This criterion of a successful run was used in all future maze tests. Right-Left Run. After the open run was mastered, the blinds (1 and 2) were placed in the runway in such a position that the animal was forced to follow a R-L pattern to reach the goal box. The glass door was always left open behind the last blind alley in this and future runs. The opposite glass door was always closed. In this R-L run and in the following phases of maze learning, the objective was to train the guinea pig that the exit to the goal box was always behind the last blind alley. The same criterion of accomplishment was used as in the open run. Left-Right Run. The blinds were reversed to a L-R position and the same criterion employed. Alternate-Sequence Run. The guinea pigs were trained to run to the left goal box or to the right goal box when the blinds were shifted in chance order. The pattern was set for either a R-L or L-R run. The chance order for setting the blinds was established by flipping a coin for two hundred times; the head side of the coin 5^ was designated as R-L, and the tail side of the coin was L-R. The order for shifting the blinds which was established by this method was also used to set the alternate runs for the retention test. Retention Test. At various intervals after the guinea pigs had learned all of the runs, they were given a retest of the random alternate sequence pattern. Litter- mates were always tested at the same time interval after original learning of maze patterns. Shock Procedure. A shock was given to insure quick departure from the starting box. However, after a few runs the normal and hypothermic-anoxic animals often left the starting box without waiting for a shock. A few of the anoxic animals left the starting box without a shock. A shock was administered if an animal entered a blind alley. No shocks were given while the animal was moving. If a guinea pig tarried longer than 120 seconds at any one point, it was given a shock to speed it on its way. Scoring Procedure. From the records kept of the maze testing, several scores were available for use as indices of the learning that had taken place. These scores were: (1) total time score, (2) total run (trial) 55 score, (3) total shock score, (h) total goal box attempt score, (5) total repeat error score, and (6) total error score. The time required for the guinea pig to enter the goal box was recorded for each run. The total time score is the sum of the run times. A total run (trial) score is the total runs neces sary for the guinea pig to learn the maze pattern. All shocks given the animal were tabulated. The total shock score was a sum of all the shocks administered. Each time the guinea pig attempted and failed to enter the goal box, after entering runway to the box, was counted as a goal box error. These errors were summed for the total goal box attempt score. A repeat error was recorded each time the guinea pig retraced its path away from the direct route to the goal box. The repeat errors were totaled for the total repeat error. An error was recorded each time the animal turned from the direct route to the proper goal box. The total error score was the sum of these errors plus the goal box attempt and repeat errors. All of the above scores were recorded in such a manner that they could be considered separately for each phase of the maze learning. CHAPTER IV RESULTS AND DISCUSSION Most of the basic raw data for each animal appear in Table II in the Appendix. Statistical evaluation and graphic presentation of these data are included with the discussion of the results which follows. Standard statistical analyses were made following methods in Underwood et alii (195^) and Moore et alii (1951)9 "significant" indicates 5 per cent probability of occurrence due to chance alone, and "highly significant," 1 per cent probability of chance occurrence. The t-test for matched groups was used (Underwood et alii, 195^). Correction was made for small samples (Moore at alii. 1951). The difference between the means of the maze performance scores was evaluated. Figure 13 shows a graphic presentation of raw data scores for total errors including errors on retention tests. This graph includes only those animals which were subjected to both the original and retention tests. As will be noted, no retention errors were made by control guinea pigs, numbers 7 and 139 and control animal number 2 made only one error on the retention test. In this graph, an effort has been made to show the relative dif ferences in total errors between anoxic, anoxic-hypothermic, 57 To+al Errors ^ c mroTfi 0*f Retention lekt And Do/^s Retained ^Anoxia Animttl f Control Animal From O th e r S o urce. r\LOorvtrol A nim al, bu6am c S o u rc e . ri-Anoxik-Hypo'^Grmic. Y Animal C”Lovuest Tempenatore. During Exposure. 0 | h Figure 13. Comparison between number of total errors for anoxic, anoxic-hypothermic and control animals Numbers at the bases of the bars indicate littermates. Note that control animal number 2 made only one error on the retention test and control animals number 7 and 13 did not make any errors on the retention test. The errors made in original learning are indicated by the lower portion of the bar, and the black upper part of the bar indicates errors made on the retention test. 58 and control guinea pigs. Littermates can be compared by- finding like numbers at the bases of the bars. Days after original maze learning and the lowest temperature during anoxic exposure are expressed above the bars. It is shown that more errors were made by the anoxic animals in learning the maze than by the anoxic-hypothermic animals. Figure 1^ shows a graphic presentation of the lowest body temperature during exposure to anoxia compared to the number of runs (trials) per minute of anoxic ex posure. It is noted that the animals with the lower temperature required fewer runs (per minute exposure in anoxia) to learn the maze. Table III, page 60, summarizes some statistics for maze scores. It will be noted that all t-values for the comparison of the anoxic and anoxic-hypothermic guinea pigs are significant or highly significant in the original maze learning except for the t-values for the comparison of shocks applied to the animals. The t-values for shocks applied during the retention test are not signifi cant. As will be pointed out in the discussion, there are other evidences that the shocks did not deter learning in any one group, but merely served as a device for expediting the maze learning. Although retention error totals are not signifi cantly different when all pairs are considered, there is ^9 0 •H TJ ;8o c d 1 0 ’ H o X •HO 170 -p 1 •H c d ( C O ! •H O ' yo dUP a * o 0 ; • U 1 —1 0 ' C O O d A ^ •H M 40 M0 _ •H f H 0 H d o d (M -H ' . a ZÛ- f 0 9 _ ff H /O' _ ID rH ■ :!k C O 1 -P a 1 25 «6 M Anoxic- i-Ljpo'lhernoïc Guiir]oatij ® Anoxic Guinea Pa Numbers Jndica+a il'iirermates . < u Î, 9. % o °s’ Lowest temperature C. during exposure to anoxia Figure 1^. Lowest body temperature compared to anoxic time-run score. Note that the anoxic-hypothermic guinea pigs made fewer runs to learn the maze during original learning if the minutes of exposure to anoxia are considered. The anoxic-hypothermic animals were also those animals which had the lowest body temperature during anoxic exposure. 60 TABLE III t FACTORS AND PROBABILITY, MAZE SCORES FOR LITTERMATES Measurement Groups compared Degrees of Freedom, t, & p Values of differences in means D.F. t P Total Errors Original Anoxic vs A-Hypothermic 11 3.18 0.01 Total Repeat Errors Anoxic vs Original A-Hypothermic 11 2.54 0.02 Total Shocks Original Anoxic vs A-Hypothermic 11 1.61 0.50 Total Time Original Anoxic vs A-Hypothermic 11 2.91 0.02 Total Time Original A-Hypothermic vs Control 5 0.92 0.50 Total Time Original Control vs Anoxic 3 0.02 Total Runs Original Anoxic vs A-Hypothermic 11 2.20 0.05 Total Runs per Minute Exposure Original Anoxic vs A-Hypothermic 9 5.39 0.01 Total Errors Retention Anoxic vs A-Hypothermic 8 2.18 0.10 Total Shocks Retention Anoxic vs A-Hypothermic 9 1.25 0.30 Total Shocks Retention Anoxic vs Control 3 0.^7 0.70 Total Shocks Retention A-Hypothermic vs Control 3 0.31 0.80 l'ûien reference is made to Table III, it should be kept in mind that the t factors were obtained on a litter- mate pair score-to-score basis, discounting the length of time the animals were exposed to anoxia. It is important that the survival time in anoxia for the anoxic-hypothermic was 38.6 per cent longer than their uncooled littermates. The cooled animals not only remained longer in the anoxia, but also made significantly better scores in learning the maze. 61 a greater increase in errors made by the anoxic animals as the time between original and retention tests increases (2, 21, 118, 1^0, and 260 days retention were considered). Retention of learning is dependent upon the time allowed to elapse between the original test and the retest. For example, if the shortest and longest retention periods are considered, the following is discovered; the dif ference in the means for the two day retention group is four errors; the difference for the 260 day group is 10^ errors. Hence, if the fact is considered that the anoxic- hypothermic newborn guinea pigs endured anoxia 38.6 per cent longer, it is apparent that hypothermia exerts some beneficial effects on asphyxiated day-old guinea pigs as far as retention of learned activities. Becker (19^6) reported that guinea pigs asphyxiated and then resuscitated at birth took "roughly twice as long to train" to run a maze. He reported that the trials, errors, and repeat errors were twice as great for the anoxic animals as for their normal littermates. In the present study, the anoxic animals showed the same poor performance as did Becker*s guinea pigs, while the anoxic- hypothermic animals were equal to or better than the performance of the control animals. In the present experiment, some of the guinea pigs did not survive to run the maze. Table III shows data for surviving littermate pairs only. All animals. 62 whether they had a surviving littermate or not, were given the maze tests. Table IV shows data for all animals which survived to run the maze. All surviving animals are compared as anoxic, anoxic-hypothermic, and control groups. These data indicate that the differences in maze scores were dependent upon some factor associated with the cooling procedure, as this condition was applied unequally to the groups drawn from the same population. As summarized in Table V, page 64-, 13 out of 25 anoxic animals survived to run the maze, 16 out of 23 anoxic-hypothermic, and 7 out of 16 control animals so survived. Regardless of the fact that the anoxic- hypothermic animals had undergone exposure to anoxia longer, they survived to maturity in greater numbers than their uncooled littermates. Body weight at birth was not correlated with body temperature at birth. However, the same environmental conditions were not maintained for all mother animals and their litters. With the available facilities, it was impossible to provide assurance that all guinea pigs would be born under the same conditions. Simple inspec tion of Figure 15, page 65, will indicate that a great variation existed in body temperatures at birth. There is a range of temperatures from 85.6^ F. to 100.4° F. (29.8° to 38.0° C.). It is believed that the birth temperature reflected the conditions present in each 63 TABLE IV MEAN SCORES FOR ORIGINAL LEARNING AND LOWEST TEMPERATURES OF ALL ANIMALS THAT SURVIVED TO RUN THE MAZE Means Anoxic Anoxic- Hypothermic Control Total of all original errors 219 102 l4l Total repeat original errors 91 53 45 Total of all original goal box attempt errors 86 56 64 Total original runs 59 42 50 Original total time to run the maze (mins.) 56.6 21.8 31.9 Lowest temperature dur ing exposure to anoxia 34.8 28.7 35.0 n n = 13 n = 17 n = 7 The temperature shown for the control animals is the mean for the lowest temperatures recorded during the first day of life. The lowest temperature shown for the anoxic and anoxic-hypothermic guinea pigs is the mean of the lowest temperatures recorded during exposure to anoxia. 64 TABLE V A COMPARISON OF THE MEANS OF SURVIVAL TIMES, LOWEST BODY TEMPERATURES, BIRTH WEIGHTS AND MORTALITY OF ANOXIC, ANOXIC-HYPOTHERMIC AND CONTROL GUINEA PIGS I Anoxic guinea pigs Anoxic-hypothermic guinea pigs 1 Control guinea pigs Lived Died Lived Died Lived Died o o 4-3 • 0 A 0 S 0 3 Ü 0 rH 0 c O > •H 0 > § C O 4 4 > !5 • 4-3 0 A a •H b b p q o o +3 • 0 A 0 a 0 c 5 0 rH 0 cd > •H 0 > B F-i *H Zi 4-3 C O 43 x i • +3 C O A a •r4 Ü0 m o o 43 . 0 A i3 ^ o 0 1 — 1 0 C d > •H 0 > a A *r4 C d 4-3 C O +3 XI • 43 to A a •rH ixO p q 0 0 43 . 0 A % % i3 Ü 0 rH 0 C d > " •H 0 > a s s C O X • 4-3 0 A a •iH t u b p q 0 0 A 0 •iH -43 p q 43 X • 4-3 0 A a •rH t u b p q 0 0 g # A 0 •rH 4-3 p q $ m a •rH t u b p q 3 4 .8 133 82.2 3 ^ .1 147 81.4 28.7 237 90.8 27.4 318 8 4 .8 35.8 104.33 35.0 1 77.9 Normal delivery Caesarean delivery Normal Caesarean delivery delivery Normal Caesarean delivery| delivery Normal delivery Caesarean delivery Normal delivery Caesarean delivery Normal delivery Caesarean delivery 10 3 4 1 8 ..... 12 4 1 2 5 7 0 6 3 "Died" indicates death sometime after the first day of life. Survival times are expressed in seconds, lowest temperatures are expressed in degrees centigrade, and birth weights are expressed in grams. As will be noted, there is no "survival time" expressed for the control animals, as they were not subjected to experimental anoxia. 65 59 + 3 f > • 35 " 34- 33- 3X- Jl Jô , 2g '#=^9 . 9 g > , 8 0 % m I ? /a "^1 ^ ' 5 9 % «8 n • 9 ? < 2 > Q ' 1 % » I > 1 1 ' 1 » 1 1 4— I 1 1 1 1 f c 1 » 5® « (iP *S 76 7S ÎO « 90 95 Ub t'r aû Us Pa /3S ih Gr^iS' Bodt# U^C/Q^f • " M Sir+K O Caesarean section Q Normal delivery Numbers indicate littermates Figure 15. Birth temperatures and weights of Caesarean and normal delivery newborn. 66 particular birth environment and that this may be evidence that newborn guinea pigs are heterothermic. It was noted that about 70 per cent of the time the last animal born in the litter had the lowest body temper ature at birth. The last animals to be born were often abandoned by their mothers, especially if the litter was large. If these abandoned newborn guinea pigs were not dried and cared for, their temperatures continued to drop until they died. In view of the findings of this experi ment and the research of previous workers on restraint hypothermia (Bartlett et alii. 1953), it is believed that possibly the restraint and trauma imposed in delayed birth causes the last born to have a lower body temperature than its littermates. It is conjectured this hypothermia could be effective as protection against anoxia accompany ing delayed birth if the animal were dried and cared for by its mother. Many of these animals survived when they were dried and rubbed vigorously and then returned to the litter. General Notes. Observations. and Comments Although the following data regarding the present study are not susceptible of statistical analysis and confirmation, it is believed they are noteworthy enough to be considered in evaluating the present study and any future study of anoxia and hypothermia of the newborn 67 guinea pig or human newborn. Observations and comments regarding the birth and early development of the guinea pig. Guinea pigs delivered by Caesarean section in this study did not have different breathing rates than those of normally delivered, full-term guinea pigs. Haddard, Hsia, and Gellis (1956) reported that breathing rates of premature and full-term infants delivered by Caesarean section are slightly higher than those for normal delivery full-term infants. Caesarean section guinea pigs must be stimulated as soon as the fetal membranes have been removed or they often die of what appears to be atelectasis. Stimulation can best be accomplished by the experimenter pulling the newborn's ears, stroking the genital area, or rubbing vigorously with a rough turkish towel. It was also observed that under conditions of natural birth, the mother guinea pig often chewed the ears of some individ uals of her litter. The animals with the chewed ears were those newborn which showed evidence of respiratory difficulties. Animals, Caesarean and normally delivered, that survived developed a characteristic "guinea pig jump." This "jump" can best be described as a hopping action in which hind limbs are moved quickly. The right rear limb was extended while the left was flexed, then the left 68 rear limb was extended while the right was flexed. This limb movement gave the guinea pig a twisting motion of the pelvic region. This "jump" appeared in healthy newborn during the first day and became less evident as the animals reached maturity. Those animals which failed to develop this characteristic invariably died. Animals that did not demonstrate a response to the "key clang" by the first day usually did not survive. (The "key clang response" was movement of ears and move ment of facial muscles when a ring of keys was jingled.) Young guinea pigs will often eat newspaper and redwood shavings if these materials are used as bedding in their cages. Many of the newborn guinea pigs that eat these materials die. Although two commercial breeders of guinea pigs stated that this was a generally known fact, this investigator could not find any references in the literature advising against the use of newspapers or redwood shavings in the newborn cages. Observations and comments regarding the •immediate and latent effects of anoxia and hypothermia. During cooling and rewarming it was noted that the facial tissues appeared to be congested and cyanotic when shivering occurred. Often the heartbeat could not be picked up on the tape recorder just before and during the shivering, and as body temperature increased the heartbeat became 69 audible again. In the preliminary cooling trials, a stethoscope was used, and it was noted that during shiver ing the heartbeats appeared to be ectopic and extremely fast. This condition was interpreted as being due to fibrillation. Rosomoff and Gilbert (1955) have suggested that shivering in dogs raises venous pressure and causes fibrillation. Perhaps their findings offer an explanation for the inability to record a heartbeat on the tape recorder during severe shivering in these present experiments. Every effort was made to place the animals in the nitrogen atmosphere while they were still being cooled; thus severe shivering was generally circumvented. Although it was observed that shivering was prevented by this means, no definite conclusions can be made concerning why this was so. It is conjectured, however, that the anoxia exerted a narcotizing effect on the hypothalmic or sympathetic functions and thus prevented shivering, ^hen shivering was prevented, the heartbeat was usually audible until asphyxia was carried to completion. In the present experiment, the anoxic animals exhibited more symptoms of cerebral edema and pressure (such as rigidity of the neck, stupor-like behavior, and slow heartbeat) during and immediately after asphyxiation than their cooled littermates exhibited. 70 It is conjectured that possibly brain volume was reduced in the guinea pigs while they were being cooled, and that this hypothermic reduction in brain volume counteracted the anoxic edema due to asphyxiation. (Perhaps it would be necessary to assume, however, that during most of the anoxic state there existed a condition of stagnant anoxia, as cerebral edema is known to exist in this type of anoxia, and not necessarily in other types.) The basis for assuming that stagnant anoxia existed in the guinea pigs in the present study was the evidence (by autopsy) of extravasation of blood and small hemorrhagic areas found in the brains of a few of the anoxic animals. As compared to some of the anoxic- hypothermic animals which died, the anoxic guinea pigs had little blood in their superficial vessels, but had congestion of such organs as the brain, liver, and spleen. Relaxed anal sphincters were present more often in the anoxic guinea pigs than in their anoxic-hypothermic littermates. The symptoms shown by the anoxic animals in this study were similar to those present in the anoxic guinea pigs studied by Windle and Becker (19^3) and are the symptoms of stagnant anoxia. Although both anoxic and anoxic-hypothermic animals in the present investigation exhibited epilepti form seizure during anoxia and recovery from anoxia, the anoxic animals' convulsions were more tonic than the 71 anoxic-hypothermic animals. Baldwin et alii (1956) have observed that chimpanzees had less epileptiform activity upon stimulation of the temporal lobes after hypothermia had been induced than prior to cooling. Since it is well known that edema of the cerebral areas may contribute to epileptiform seizures, it is possible that the differences in types of seizures in the cooled and uncooled guinea pigs in the present study were due in part to the effect of hypothermia in reducing brain edema. By inspection of Figure 16, it is possible to determine that there was no definite relationship between duration of asphyxia time and resuscitation time, although Windle and Becker (19^3) found that resuscitation time with their animals was roughly proportional to the duration of asphyxia. It should be emphasized, however, that Windle and Becker asphyxiated their guinea pigs in utero, and in this study the guinea pigs were asphyxiated in a nitrogen atmosphere during their first day of life. Windle and Becker resuscitated their animals with oxygen in a small rubber bag while a modified mouth-to-mouth resuscitation was used in the present experiment. The carbon-dioxide present in the resuscitation method in the present study could have greatly influenced recovery time. In the present study, guinea pigs which were cooled the most exhibited the longest time for their temperature to return to the starting temperature. 72 X § C C O 0 a CO o X 0 W 0 d d •H S- 7- 6 6 4 5 2. I / h < hO » # m i ■ ( f # • T ■ f f Ar\o)t»C’H*jpo+Acfm'‘ e'' fc«r\ple<e A«coveru • T V * j □ a Arvm'G- - U l f t d . • a A f N o ' t f lO -Complete, Recovers O « A n » **6 - D ie j, » 0./ A6 si" a7 48 AS f . ù Minutes for resuscitation (first gasp) Figure 16. Exposure time and resuscitation time. Resuscitation time was determined as the time interval between the last gasp during exposure to anoxia and the first gasp after anoxic exposure. This was the time required to revive the animal via artificial respiration. 73 Miller et alii (195^) noted that animals cooled the most exhibited the longest recovery time. In this present experiment, judgment of recovery was based on the return of body temperature to the level present at the start of the anoxic or anoxic-hypothermic procedure. Miller's criterion for complete or nearly complete recovery time was the return of voluntary move ments and the ability to progress by the movement of all four legs. The animals in this study passed through the same stages described by Miller, but they reached the "crawling stage" long before their temperatures returned to normal. Animals in the present study still displayed slow reflex actions while Miller's criteria indicated complete recovery. However, it must be noted that Miller used 95 per cent nitrogen with 5 per cent carbon dioxide for asphyxiation. Recovery in the present experiments also involved recovery sufficient to allow the guinea pig to live to maturity so that it could be tested in maze activity. Miller was apparently interested in immediate recovery and not how long the animal was to survive after the initial stages of recovery. An examination of the raw data presented in Table II in the Appendix will show that there is a great difference between the degree of recovery sufficient to appear grossly nfrmal immediately after anoxic exposure and recovery sufficient to live to 7^ maturity. Most animals left in the nitrogen chamber after the "last gasp" in this experiment died by the seventh or eighth day, and all of them died by the tenth day after exposure. Autopsy findings on the animals which died after overexposure showed some of the animals had extremely enlarged gall bladders, as large as 19 millimeters in diameter. Petechial areas were present in the temporal lobes of the brain and congestion of the lungs was present. In many cases, the lungs appeared to have saccules filled with a semi-solid, red gelatinous substance. The lower lobes appeared to be consolidated. Although the present writer is not especially trained in pathology, these findings suggested a type of consolidation he has seen in human lobar pneumonia. At first thought, it was believed that the artificial respiration technique might have been a causative factor in the death of these animals; however, the time and effort necessary to revive these "over exposed" guinea pigs was not different from that for the animals which survived. Possibly some of the animals which survived also had lung pathology of the type described, but survived because they had suffered less cerebral damage from the anoxia. In the present study, motor and sensory disturb ances were evidenced by several different syndromes in the animals which survived to run the maze. Animals which 75 made the most errors of all types exhibited these syndromes to the greatest degree. Such animals often coughed and choked when they ate lettuce or other greens, indicating disturbance of sensory areas eliciting the swallowing reflex. The low error group seldom coughed or choked. Response to light, sound, and heat was impaired. Animals (both anoxic and anoxic-hypothermic) that made more errors had occasional tics or twitchings of the facial muscles when they encountered a blind alley from which they could not readily find the exit. It has been previously shown in the statistical analysis that the highest error group was that of the anoxic newborn. Although both the cooled and uncooled newborn guinea pigs showed varying degrees of convulsive twitchings, torti collis, uncoordinated movements, and comatose states during immediate recovery from anoxia, most cooled animals recovered sufficiently so these symptoms did not reappear under stress in the maze, while the anoxic animals did not recover. Although the technique of asphyxiation varied from that of Windle and Becker (19^3), all animals seem to show the same symptoms Windle and Becker observed in their guinea pigs. In both studies, the anoxic animals exhibited certain spastic and convulsive movements, abnormalities in swallowing and unresponsiveness to bright lights and sharp noises. Syndromes which were exhibited 76 by the anoxic guinea pigs but not by the anoxic-hypothermic guinea pigs in the present study lend weight to the belief that cooling tends to prevent neurological damage during asphyxia. Observations and comments regarding behavior during maze testing. One difference among the anoxic, anoxic- hypothermic, and control animals was the fact that the anoxic animals urinated and defecated more often while in the maze or when handled. An activity not observed in the control animals and in only one anoxic-hypothermic animal was that under stress of "apparent indecision" in the maze, the anoxic animals would often defecate, then turn around and eat their feces. In the present study, there was random voiding and elimination by all three groups of animals (anoxic, anoxic-hypothermic, and control animals). Binder and Thompson (1953) reported that the occurrence of a trial- to-trial decrease of elimination rather than random elimination was useful as a measure of fearfulness in rats. A review of the literature did not reveal any similar results for guinea pigs. If this criterion is applied to guinea pigs in the present maze learning experiment, no differences in fearfulness of the maze existed for the three groups, anoxic, anoxic-hypothermic, and control animals. As was emphasized previously, no significant 77 difference existed in the number of shocks administered. It is evident that neither fearfulness of the maze or shocking procedure affected learning adversely in any one group. Animals (both anoxic and anoxic-hypothermic) that made fewer errors had a tendency to "burt" (a low gut tural intermittent noise) when they had solved the maze problem and had reached the carrots without too much effort. These noises were similar to those made by normal animals when they were underfed or fed on an inadequate diet, and then offered carrots, green alfalfa, or some other "attractive food." Although the slower-to-learn guinea pigs ate the carrots, they did not eat as voraciously or make these sounds as often. The more adept the animals were at learning the maze, the more obvious their responses were to food and safety of the goal box. Those animals which presented the most evidence of neurological damage during recovery from anoxia, such as holding their heads to one side (usually the left), were those animals which failed to follow what Woodworth (1938) termed the goal gradient. This gradient hypothesis is based on the findings of various workers that if all other factors are equal, blind alleys will be eliminated in reverse order, beginning with the blind alley closest to the goal box. 78 A study of the individual run patterns indicates that when the anoxic-hypothermic or control animals made errors at the first blind alley, it was because they appeared to be exploring the area or retracing their steps to get some sort of clue from the setting of the first blind alley. Woodworth (1938) described this type of activity with rats and indicated that retracing to the entrance does not mean lack of orientation, but rather that this particular part of the maze is familiar. Most of the animals which showed symptoms of neurological trauma made erratic entrances into the blind alleys and did not retrace their path to "familiar" areas of the maze. Considering place learning, it is of interest not only what parts of the maze the guinea pigs learned first, but what errors were eliminated during adaptation to the maze. The more adept guinea pigs made use of intermediate goals or landmarks in their early progression toward the safety of the goal box. The less adept guinea pigs were not as quick to make use of intermediate goals. The traumatized anoxic animals had difficulty in learning the relationship of the first blind setting to the remainder of the blinds. The animals with less ability to learn the maze were most affected by the direction of departure from the starting box. These animals seemed to be affected a 79 great deal by what Schneirla (1929) called "centrifugal swing"— the tendency of a rapidly moving animal to be brought into a blind alley partially because of momentum and motor convenience rather than for any other reason. Various functions involving spatial elements were impaired in the most severely traumatized anoxic guinea pigs. Petechial areas found in the temporal lobe region of the anoxic animals that died may be associated with this impairment. It is commonly stated in physiology texts (Fulton, 19^7) that ablation or trauma to the temporal lobes or fibers from the temporal lobes some times causes inability to recognize objects visually, inability to interpret point-to-point relationships, and inadequate localization of stimuli. Numerous references can be found in the psycho logical literature which present results from experiments with animals deprived of portions of their cerebral cortex. Humans or animals with damaged brains often exhibit lack of flexibility in their mode of attack of a problem, while their normal colleagues are flexible, less stereotyped, and show variability in their behavior. The less adept anoxic animals in this study exhibited definite stereotypy, rigidity, and confusion during the earlier stages of learning the maze problem. When an animal followed the same wrong pathway repeatedly, it was considered as being stereotyped in its method of 80 attack. The repeat errors were also a measure of stereotypy. Rigidity was considered to be present when an animal found the correct goal, but was slow to eliminate errors, especially the goal box attempt errors. Becker (1946) states that stereotypy of behavior is not common in the guinea pig, but is common in the laboratory rat. Anoxic guinea pigs were noted to be quite thigmo- tropic when inside a blind alley. They tried to follow along the wall of the blind alley in order to find an exit. The less traumatized animals showed this tendency to a lesser degree. Guinea pigs are not normally thigmo- tropic, while rats are thigmotropic*. Responses to various sound frequencies. As was previously mentioned, testing with tuning forks indicated loss of response to certain frequencies in some guinea pigs, and further testing was done with a tape recorder. Figure 17 indicates the responses to frequencies by each group, anoxic, anoxic-hypothermic, and control animals. The control animals * responses were recorded for each frequency and the other two groups* responses graphically represented in arbitrary volume units above or below the normal animals* responses. Normal guinea pig response is represented as a straight line, and less or greater than normal response is charted above or below this straight line. This graphic form of showing hearing loss is used 81 ô+ Vôiumtv #—e AnoifjC f < 0 3Z [ig 7 J S Ù /ô# »40 /4o% FreQoenccj c p *. Figure 17. A comparison of phonoreception in anoxie, anoxic-hypothermic and normal (control) guinea pigs. The curves represent the mean responses of each group. Normal (control) guinea pigs’ responses are plotted as a straight line. Responses greater than normal or less than normal are shown by points above or below this straight line. The volume units shown were empiric positions on the tape recorder volume control. 82 in human audiograms. Loss of phonoreception was noted as a post- asphyxiation syndrome in many of the anoxic animals. Most of the anoxic-hypothermic and control animals did not show this loss of response to sound. Windle, Becker, and Weil (1944) found that some of their anoxic animals failed to respond to sharp noises which may have been due to the observed extensive nerve cell loss in sensory areas such as the thalamic nuclei and geniculate bodies. In human beings, it is known that most auditory fibers destined to reach the cerebral cortex synapse in the medial geniculate body which is situated in the posterior portion of the thalamus at the same level as the lateral geniculate body. Guyton (1958) states that most of the qualities of sound are possibly perceived by the nuclei of the medial geniculate body or associated nuclei of the thalamus. The ability to perceive sound, especially in the low tones, was present in the hypothermic guinea pigs and absent in the uncooled littermates. Stevens et alii (1935) and Tasaki and Davis (1955) constructed a graphic representation of frequency responses related to cochlear areas; the map of cochlear responses for man was deduced from the microphonie responses in the guinea pig. Prosser (1950) states that men and guinea pigs are sensitive to vibrations of about the same frequency range, but the guinea pig has one additional turn to the 83 cochlear spiral. Considering the similarity of the response mechanism of the guinea pig and man, it is reasonable to suggest that conditions causing phonorecep tion loss in the guinea pig could cause similar conditions in man and vice versa. Myklebust (1954) believes that anoxia at birth is one cause of deafness in children. General observations concerning colonial living. After the experiment was carried to completion, the experimental animals were allowed to breed in a colony cage. The anoxic animals were definitely inferior in competition with the other animals. Most of the anoxic females did not care for their young as well as the control or anoxic-hypothermic animals. For example, in a colony cage, the normal mother guinea pig would back off in a corner with her litter behind her when danger threatened, but the anoxic mother would allow her young to be out in the center of the cage where other animals would bite and step on them. In the present study, although it was impossible for an independent human observer (trained in the breeding and care of guinea pigs) to pick the anoxic from the anoxic-hypothermic or control animals, in the colony cage living, the anoxic guinea pigs were soon discovered by their colleagues. When new members were added to the cage, when competition became keen for females, or when some unusual circumstances arose (such 84 as when the animals were fed a little off schedule), the whole colony went through a process of eliminating or subduing of the most inept members. The guinea pigs engaged in biting and picking patches of hair (sometimes to the extent of complete baldness) from the unfortunate underlings. The most often subdued members of the colony were the anoxic guinea pigs with poor maze learning records. Windle and Becker (1943) found that it was quite impossible after two or three weeks to distinguish between some of the anoxic damaged guinea pigs and their normal littermates as far as general behavior was con cerned, although pathological changes were found in the brains of the anoxic animals. Comparative studies. It must be borne in mind that the results reported in this investigation were ob tained from guinea pig experimentations. Whether the same changes in behavior occur in anoxic and anoxic- hypothermic human newborn is another problem for investi gation. The ill effects of hypothermia should be thoroughly investigated to determine if these effects outweigh the benefits of hypothermia. As was mentioned in the literature survey of this topic, retroactive studies on children showing learning problems have indicated high correlation between a history of asphyxia at birth and neurological difficulties. 85 It seems possible, and it is suggested, that the main differences between the results obtained in this study and any investigation of infants would fall largely into the category of genus and species differences con cerning the limits of cooling, speed of rewarming, degree of neurological changes, and other factors which it appears would vary quantitatively rather than qualita tively. CHAPTER V SUMMARY AND CONCLUSIONS Summary 1. The ability of hypothermia to prevent neurological trauma in asphyxiated newborn guinea pigs was investigated. Normal delivery and Caesarean section guinea pigs were used in the study. 2. The split-litter technique was used with animals divided in three main groups : 1) anoxic animals subjected to asphyxia in a nitrogen atmosphere without being experimentally cooled, 2) anoxic-hypothermic animals exposed to asphyxia after they had been cooled with 95 per cent ethyl alcohol and a fan, and 3) control animals which were not exposed to either anoxia or hypo thermia. Anoxic and hypothermic procedures were done within the first day after birth. 3. All animals were observed for normal reactions. Abnormalities of behavior were recorded and the three groups of littermates compared. Specific tests were made at suitable intervals to determine evidences of neuro logical trauma. 4. The anoxic group of animals exhibited varying degrees of spastic conditions, uncoordination, hearing loss and general inability to respond adequately to their 87 environment. These neurological symptoms were observed in only a few of the cooled anoxic animals. 5. Animals that died were examined by autopsy. Gross examination revealed hemorrhagic areas in the brains, consolidation in the lungs, and enlarged gall bladders. The damage was more extensive in the anoxic animals than it was in their anoxic-hypothermic litter mates. Some of the pathological tissues were saved for a future histological study. 6. Surviving mature guinea pigs were tested for the ability to learn and retain the learning of a maze problem. Maze scores were compared and the cooled asphyxiated guinea pigs were approximately two times more adept at learning the maze than their uncooled litter mates. 7. The differences between the means of the maze performance scores for the two groups of guinea pigs, anoxic and anoxic-hypothermic, were compared by the t- test. The t factors were highly significant in the original learning for the following ; total errors and total runs per minute exposure to anoxia. Significant t- values for the anoxic and anoxic-hypothermic animals in original learning were for the following scores: total repeat errors and total time to learn the maze. In evaluating the retention of learning, the t-value factors for total errors were not significant. 88 For no phase of the maze learning were the t-values significant for the shocks applied. The t-value was significant for the time scores of the anoxic and control animals, but not significant for the anoxic and anoxic- hypothermic animals. 8. This difference in maze performance was ac complished by the hypothermic asphyxiated newborn guinea pigs regardless of the fact that they experienced as a group a 38.6 per cent longer exposure time in the asphyxiating nitrogen atmosphere. Conclusions The conclusions which can be made from this study are ; 1. Anoxia damages tissue of the central nervous system of the newborn guinea pig. 2. Hypothermia is beneficial in minimizing anoxic trauma in the asphyxiated newborn guinea pig. 3. The marked differences in maze learning abilities of anoxic and anoxic-hypothermic guinea pig littermates indicate that hypothermia prevents or mini mizes changes which would tend to produce poor learning. 4. Hypothermia applied to anoxic day old guinea pigs prevents hearing loss which would otherwise occur if the anoxic animals had not been cooled. LITERATURE CITED LITERATURE CITED Adolph, E. F. 1951. Responses to hypothermia in several snecies of infant mammals. American Journal of Physiology. 1^:75-91. Alexander, M. L. 1945. The treatment of shock from prolonged exposure to. cold, especially in water. Office of Publications Board Department of Com merce, Washington, D. C.: Report Number 250. Allen, J. 1904. The associative processes in the guinea pig. Journal of Comparative Neurology. 14:293-359. Anonymous. 1937. Report of the Committee on Asphyxia of The American Medical Association. 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Henry Holt and Company, New York. 889 pp. Yant, W. P., J. Chornyak, H. H. Schrenk, F. A. Patty, and R. R. Sayers. 1934. Studies in asphyxia. United States Public Health Bulletin, 211:4o. Yapp, W. B. 1939. An Introduction to Animal Physiology. Oxford, at the Clarendon Press. 319 pp. APPENDIX Ra/W Oa*W/ -from Evpsrimsnf M m OPEhJ-WN lVlGHT-LEf=r-RUN/ LEFT-f?JGHT-gW ï î l a s î ^<3 (o S Z 3/Ù £JiûÜlsd o4 fUd<r ibcaien ItLâùÊA Turjcd l < 1 f S I in in L»,& Z < ! » L „ fuhi^s jnmihL 0.iS3n HÀ â f f i [ XciL^àkJ^l inhtrs J#w y Tubitksl ic-fardr id e a v)po^4ic Lç/ net^p tn-hes % ? u/rthcr û-f j / < y / | . A t & O À d Ù L “ . L u n û û J 4 t t r ca4UL&iL to l Êi kJt ei r_5»AsWv: ~ .kawjr 'O r- gy/a / 4 f ï L i i x n 77 /cTiJA ■Frointo »?j ; f | ( D , ^ / @|4Zj2 Jf ? AT 3 A M »3 f'J 7 O M w : J L i fs m o Q M 1L% i'€€orci\ \Vtli1C< ^K'ôma ànnéÂ4JtànêL m ■ a P r io r r^i-- J e s . iUliiai àHi3o eioj! 69 i WjvS T A B L E 3 1 ., Cônlinued W trr-HOhl LP-FT-MOar 4LTEI^WATE-KUA/ WHO U S ! ? 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Creator Carpenter, Dale Leonard (author)

Core Title The ability of hypothermia to protect newborn guinea pigs from anoxic trauma

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Degree Master of Arts

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Permanent Link (DOI) https://doi.org/10.25549/usctheses-c39-278903

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University of Southern California Dissertations and Theses