Auditory Evoked Potentials and Auditory Behavior Following Prenatal and Perinatal Asphyxia in Rhesus Monkeys ALLAN F. MIRSKY MERLE M. ORREN LINDA STANTON BARBARA C. FULLERTON SANDRA HARRIS Laboratory of Neuropsychology Division o f Psychiatry and Department of Neurology Boston University School of Medicine Boston, Massachusetts RONALD E. MYERS Laboratory of Perinatal Physiology NINCDS, National Institutes of Health TWOtypes of asphyxia were studied in monkeys, total asphyxia during mid-pregnancy (94-98 days gestation) and combined partial and total asphyxia at term (165 days gestation). Auditory evoked potentials and the acquisition of 2 auditory discrimination tasks were studied in asphyxiated animals as well as in a group of controls. The brains of all asphyxiates were examined histologically. N o auditory discrimination deficit was found in the asphyxiated animals; however, the auditory evoked potentials differentiated between control and asphyxiated animals, especially those with verified inferior colliculus damage.
Over 30 years ago, W. F. Windle published the first in a series of papers describing the effects of asphyxia neonatorum in guinea pigs and monkeys (Faro & Windle, 1969; Ranck & Windle, 1959; Windle, Becker, & Weil, 1944). In the monkey, total asphyxia at birth results in neuropathological changes in the brain stem, thalamus, basal ganglia, and cerebellum (Ranck & Windle, 1959). Particularly affected are the inferior colliculi, but damage is also incurred by other sensory relay nuclei of the auditory, somesthetic, and vestibular pathways. Myers (1967) has confirmed these findings and, in addition, shown that vulnerability to the effects of total asphyxia in the monkey is related to
Reprint requests should be sent to Allan F. Mirsky, Laboratory of Neuropsychology, Division of Psychiatry and Department of Neurology, Boston University School of Medicine, 80 E. Concord Street, Boston, Mass. 02118, U.S.A. Received for publication 7 April 1977 Revised for publication 28 April 1978 Developmental Psychobiology, 12(4):369-379 (1979) Q 1979 by John Wiley & Sons, Inc.
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gestational age (Myers, 1971a). Thus, fetuses of 90-95 days can withstand up to 30 min of cord clamp without sustaining detectable pathology, whereas only 10 niin in the term fetus (165 days) is sufficient to inflict serious damage. Myers and his colleagues have also developed alternative methods for inducing pre- arid perinatal asphyxia in monkeys which may model more closely the events and consequences of :I) difficult labor and delivery (Adamsons, Mueller-Heubach, & Myers, 1971 ; Brann 8c Myers, 1975; Myers, 1969, 1971b; Myers, Mueller-Heubach, & Adamsons, 1973). These procedures, involving a prolonged period of partial asphyxia, result in neuropathology of the cortex and basal ganglia. The functional consequences of pre- and perinatal asphyxia, as might be measured behaviorally or electrophysiologically, have received only limited experimental attention. Monkeys subjected to total asphyxia at birth show a wide variety of permanent and transient behavioral deficits on tasks of delayed response, unsignalled avoidance, general reactivity, visual discrimination, visually guided behavior, and loconiotor development (Hyman, Berman, & Berman, 197 1; Hyman, Parker, Berman. 81 Berman, 1970; Sechzer, 1969; Sechzer, Faro, & Windle, 1973). In light of the vulnerability of the auditory system to total asphyxia at birth, it is surprising that only one investigation has been concerned with subsequent auditory behavior. Using an avoiclance paradigm Berman, Karalitzky, and Berman (1971) found an increase in auditory thresholds of monkeys asphyxiated at birth. The present work provides some preliminary behavioral and electrophysiological observations on auditory functioning in monkeys subjected prenatally or at term to total and/or partial asphyxia.
Methods Asphyxiations The asphyxiations were carried out while the fetuses (Mucaca vnulatfa) remained in the uterus. The partial asphyxia of 5 term fetuses was accomplished by constricting tlie maternal aorta (Myers et al., 1973) or by administering carbon monoxide t o the mother (Ginsberg & Myers, 1976). In a single instance, the asphyxia resulted from the procedures (catheterization of the mother) used to prepare the animal for study. In 3 animals the partial asphyxia was followed by a period of total asphyxia (ranging froin 1-15 min) brought about by clamping the umbilical cord. The magnitude of asphyxia was monitored and controlled after catheterization of the fetal femoral artery by measuring blood pH, pO2, and pCOz (Brann & Myers, 1975). In the 3 mid-pregnancy (94-98 day gestational age) asphyxiations, the umbilical cord was exposed through a small incision into the uterus and tightly occluded manually for a period of 30-35 min. This duration was selected on a basis of prior study (Myers, 1971a) to be sufficient i:o produce damage to structures in the brain stem. At the end of this time, the umbilical cord (or the heart) was injected with epinephrine and gentle massage of the heart was instituted through the fetal chest wall to restore circulation and heart beat. The various incisions were closed in anatomical layers and the pregnancy allowed to continue until term.
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Acquisition, Housing and Rearing Conditions The mid-pregnancy animals and 2 of the controls (C-M and C-C) remained with their mothers for 3 weeks after birth. Thereafter, they were separated from the mothers and hand-reared until they were able to feed themselves independently, usually at 2-3 months of age. All of the term animals were hand-reared from the time of birth. Of the remaining control animals, C-L was hand-reared from the age of 1 week whereas C-W, a feral animal, was acquired for the present study at the age of 2 years. All animals were housed in individual cages in the busy and active general monkey housing area. Except for C-W, all were subjected to large amounts of individual handling from an early age.
Experiment I: Auditory Discriminations Subjects The subjects were 5 monkeys asphyxiated at term and 3 unasphyxiated control monkeys. All were 16-18 months of age at the beginning of training except for 1 control (C-W) which was 1 year older.
Testing and Training Procedures During training, the monkeys were water deprived; monkey chow was available ad libitum. Animals were tested while seated in a small restraining chair in a testing chamber (105 cm high x 44.5 cm wide x 53.3 cm deep). The combined manipulanda-discriminanda were a triangular array of switches mounted on the wall facing the animal. Reinforcement was a drop of commercial orange-flavored sugar drink delivered through a tube near the mouth. The animals began training, by successive approximations, on a self-paced auditory discrimination task (Dewson & Burlingame, 1974) in which they were to press for 2 sec the top center switch of the triangular array. The 2-sec hold resulted in an auditory stimulus presented through a center-mounted speaker. A discriminative response involving the left or right switch was then required depending on the auditory stimulus. In 1 version of the task, the monkeys were reinforced for pressing the left switch for white noise or the right switch for a 2500-Hz tone. In the 2nd version, a discrimination between 2 tones (2500 vs 4800 Hz) was required. After an error, the same discriminative stimulus was presented on succeeding trials until a correct response was made. The animals worked for 100 trials at the beginning and 200 trials later in training. Visual cues (i.e., illuminating the “correct” switch) were used during shaping to help establish the auditory discrimination and also later in training as prompts. Eventually the animals reached a criterion level of at least 80% correct responding without visual cueing.
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Results Two term animals (T-Z and T-C) were so severely impaired in motor capacity that they could not be tested for auditory discrimination. The remaining asphyxiates showed no deficits in auditory discrimination learning and, in fact, were superior as a group to the controls in acquisition up to the level of 80% correct responses. Two of the 3 asphyxiates also reached more stringent criterion levels more rapidly than any of the control animals (see Table 1). On the tone-tone discrimination task, none of the animals in either the control or aphyxiated groups achieved levels of correct responding greater than 78% despite extensive training. Group differences were not evident on this task.’ We add here some behavioral observations made in the course of testing thcse animals. The auditory discriminations were difficult for the animals to learn, requiring in some cases up to 8 months of training. The incidence of “troublesome’’ behavior or screaming “fits,” especially when frustrated, was high among control animals. At times: testing had to be suspended temporarily. Only 1 asphyxiated animal displayed such behavior, but to a lesser degree which did not require an interruption of testing.
Experiment 11: Auditory Evoked Potentials (AEP’s) Subjects The subjects were 5 monkeys asphyxiated at term, 3 monkeys asphyxiated in mid-pregnancy, and 3 unasphyxiated controls. One monkey was tested on a single occasion at 6 months of age and the rest were tested several times between the ages o:f 14 and 36 months.
Procedure The animals were tested in a restraining chair fitted with head restraints. The EEG was recorded using a Grass Model 7 polygraph and a Technical Measurements
TABLE I . Noise-Tone Discrimination: Number of Testing Days (200 TrialslDay) to Reach Given Level.
Controls
Term Animals
Animal
70%”
75%a
80%a
85%
90%
C-M
35
41
61
66
70
C-L
35
35
37
57
58
c-w
20
25
28
30
35
T-R
18
19
27
44
47
T-E
16
18
22
28
29
T-S
7
8
11
17
18
aPerformance levels at which no overlap existed between control and term animals.
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Corporation FM magnetic tape recorder for later processing on a LINC 8 computer. The recordings were made with subdermal electrodes inserted into the scalp over the frontal, anterior temporal, and occipital regions bilaterally and over the central vertex. An additional midfrontal electrode served as ground. Auditory stimuli, presented via .03-W, 8-52 earphones affixed to the head restraints, were produced by a GrasonStadler model 901 B noise generator. The generator output was put through a 10-msec time gate, and the signals produced were presented binaurally at 2 intensities (72 and 82 dB as estimated with a General Radio sound level meter) and at 2 rates (l/sec and 1/2 sec). The recordings (300 stimuli in each condition) were made at full room illumination. The animals invariably remained fully alert throughout the recording period. The AEP analysis was performed on the vertex to the left temporal and vertex to the right temporal leads. Although recordings were made at a number of ages, only those from around 25 months of age (nearest the completion of the auditory discrimination testing) are presented.
Results The AEP's of asphyxiates differed from those of controls (see Fig. 1) along 1 or more of the following dimensions: (a) morphology; (b) latency and side differences; (c) amplitude. (a) Morphology: The AEP's of asphyxiated animals often lacked the pronounced P2-N3 waves apparent in the responses of control animals. Some of the asphyxiates (T-S and M-E in Fig. 1) showed an additional negative wave following the N2 complex that was rarely present in control animals. Pooling the 2 intensities of click and 2 rates of click presentation showed this component to be present significantly more often in asphyxiates than in controls (X' test, p < .001). (b) Latency and side differences: In control animals, component N1 peaked at 15-25 msec after the click. Differences in the latency of this component recorded from the left vs right side of the head of a given animal never exceeded 5 msec. The latency of this component was either not affected or increased slightly with an increase in stimulus intensity from 72 to 82 dB. In the term asphyxiates, the latency range for this component was 10-30 msec with the shortest and longest latencies attributable to animals with inferior colliculus damage. These monkeys differed most from the controls at the lower intensity (72 dB) and slower presentation rate (1/2 sec). One of the animals with inferior colliculus damage showed differences in the latency of the N1 components on the left vs right side of the head as great as 20 msec. In the mid-pregnancy group, the latency of N1 ranged from 10 to 35 msec, both extremes occurring at the higher click intensity. Side differences were considerable, though not always consistent, with 10 to 15 msec discrepancies for each of the mid-pregnancy animals under one or more stimulus conditions. Whereas the latency of N1 was either increased or unaffected by stimulus intensity for controls, 4 of the 7 asphyxiates, including 1 animal with inferior colliculus damage, showed a decrease in N1 latency with an increase in stimulus intensity. Component P1 was the 1st positive component which, in control animals, peaked at 40 to 50 msec. Side differences did not exceed 5 msec. Among the 3 controls, the
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AU DlTORY EVOKED POTENTIALS
ASPHYXIATES
*T-C
INFERIOR COLLlCULUS
Fig. 1. Auditory evoked potentials (AEP’s) to an 8 2 d B 1/2-sec click sourccs for all animals in the study. Also included are AEP’s from T-C, a term asphyxiate with extensive brain damagt:, including bilateral destruction of the inferior colliculi. The total sweep time depicted is 425 msec; in each case the wave form represents an average of 200-300 samples. The latcncies in msec scen in normal animals for the several pcaks shown at thc top of the figure are as follows: N 1 : 15-25; P I : 40-50; N2a: 60-75; N2b: 85-320. Note the relatively flat AEP’s for the 3 animals with inferior colliculus Icsions: T-R, T-Z, and T C and for M-A; and the generally reduced amplitudes of all asphyxiates. The text describes other differences among the groups.
latency of the components was not uniformly affected by either rate or intcnsity changes. The term asphyxiates with demonstrable brain damage (T-S, T-K, T-Z: T-C), when compared to the control animals, tended t o show a slightly slower (not exceeding 5 msec) P1 component at the lower stiinulus intensity (72 dB) and faster rate of presentation ( l / l sec). Under other stimulus conditions the P1 latencies of these animals were comparable to the controls. One mid-pregnancy asphyxiate, M-A, showed a substantial side difference in addition to a grossly deviant P1 latency, with a range of 15-25 msec on the right side and 35-45 msec on the left. The pattern of deviation was also apparent for this animal when considering the N2a component. (c) Amplitude: By far the most striking feature of the AEP’s of asphyxiates was their small amplitude compared to control animals. This is apparent in Figure 1, showing the response to 82-dB clicks presented at 1/2 sec, but was also a feature (of responses at lower click intensities and faster rates.
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The amplitude between P1 and the large peak of the N2 complex completely separates all asphyxiates from the three controls. The lowest amplitudes for each of the AEP components were consistently attributable to the term animals with inferior colliculus damage and to mid-pregnancy asphyxiate, M-A. This animal also showed grossly deviant latencies. In many instances the highest amplitude shown by an asphyxiate did not exceed or, in some cases, even come close to, the lowest amplitude shown by a control. The possibility that the extremely low amplitude AEP’s on the part of some of the asphyxiates (T-Z, T-R, and M-A) was secondary to or correlated with low amplitude, high frequency EEG was tested in these 3 cases by a computerized frequency and amplitude analysis (Mirsky, Tecce, Harman, & Oshima, 1975) of the resting EEG recorded prior to EP testing using the same electrode ptacements as in the AEP analysis. The results indicated that the asphyxiates generating the extremely small AEP’s did not show more low amplitude fast activity than controls. The small amplitude AEP’s on the part of these 3 asphyxiates (T-R, T-Z, M-A) were specific to the auditory modality and not due to the procedural flaws or a general dysfunction which might have affected equally EP’s recorded in all modalities. Two of the 3 controls and 2 of the 5 asphyxiates showing small AEP’s were tested during the same recording session for visual evoked potentials (VEP’s) to photic stimulation, These data indicated that the VEP’s of the asphyxiates were comparable to or even slightly larger than the VEP’s of the controls. Consequently, the degradation of the AEP appears to be a modality-specific effect. Tests at other ages gave the same AEP separation among animals as that presented here.
Histological Examination of Brains The brains of all term and mid-pregnancy asphyxiates were examined grossly and microscopically. No abnormality was seen in the brains of any of the mid-pregnancy animals or of term animal T-E. Definite pathologic changes were seen in the brains of the remaining 4 term animals. Animal T-S exhibited enlargement of the occipital horn of the right lateral ventricle with thinning of the overlying occipital cortex. Animal T-R showed marked nerve cell loss and gliosis in multiple thalamic nuclei including the lateral part of the posterior ventral, and the intralaminar and the lateral parts of the dorsomedial nuclei. In addition, T-R exhibited marked gliotic scars in the inferior colliculus (Fig. 2) and both parts of the superior olivary nuclei. (The normal-appearing inferior colliculus of M-A is shown in Fig. 2, for purposes of comparison with T-R.) The most marked damage was seen in T-Z and T-C. T-Z’s brain showed symmetrical atrophic sclerosis of the postcentral gyri (arm and leg areas), sclerosis and major tissue loss of the putamen and portions of the caudate nucleus and globus pallidus, and scarring of the posterior ventral and centromedial nuclei of the thalamus bilaterally. The inferior colliculi also showed great reduction in size and central scarring. Animal T-C showed a true microcephaly with a major reduction of brain size. Neurological examination of these animals at the time of sacrifice revealed the following abnormalities: Animal T-C was in a permanent state of opisthotonus and required complete hand care. Animal T-Z showed great clumsiness in all actions (more so in the arms than legs) with a scissors gait and increased adductor tone. Its placing and hopping reactions were greatly depressed although both the hands and feet showed grasping reactions. It gave some suggestion of intention tremor and past-pointing.
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(b) Fig. 2. (a) Microphotograph of the brain stem of the term animal T-R showing gliosis and absence of cells in the inferior colliculus; (b) microphotograph of the brain stem of the mid-pregnancy asphyxiate M-A showing apparently normal inferior colliculus.
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Animal T-S exhibited an abnormal posture of the hind legs in sitting, standing and walking. It was observed to walk on his heels, rather than on his toes. It showed some suggestion of a broad base in the hands. Animal T-R manifested some abnormal placement of its hind limbs, and a suggestion of this in the forelimbs. The other animals showed no neurological abnormalities.
Discussion The present investigation was undertaken as a pilot study and, as such, suffers several limitations. The combination of partial and total asphyxia of the term fetuses did not allow us to separate the functional consequences of the 2 procedures. The auditory discrimination tasks were apparently not entirely suited for our monkey subjects since all animals required intensive and extensive training. Finally, only a few animals in each condition were studied. Nevertheless, the results have raised some interesting questions concerning the relationship among behavioral, electrophysiological and neuroanatomical variables in this area of research. Although previous investigations have demonstrated a multiplicity of behavioral impairments on the part of asphyxiated monkeys, we failed to find any evidence of deficit in auditory discrimination in our asphyxiated animals. In fact, the term group, up to the late stages of acquisition, learned the noise-tone discrimination more rapidly than control animals. However, all animals had considerable difficulty in acquiring this task (i.e., up to 8 months of training). Thus, the procedure used might be more a measure of tolerance of frustration/failure than a measure of auditory discrimination ability.2 Two of the control animals were often difficult to test, especially after repeated failure to achieve mastery. This intolerance was less often observed with the asphyxiated animals and suggests the possibility of motivational basis for their superiority. The electrophysiological findings of pathological evoked potentials in the auditory but not the visual modality in the asphyxiates suggest several possibilities. All asphyxiates, regardless of whether damage to the auditory system could be demonstrated, showed AEP’s of overall smaller amplitude and lacking those late components which were often prominent in normals. Many asphyxiates also showed deviant latencies and grossly asynchronous responses from the 2 sides of the head. The amplitude measures of individual AEP components clearly separated animals with inferior colliculus damage from other animals. Casseday and Neff (1975) have reported a similar finding in adult cats with surgical lesions of the inferior colliculus. However, the same measures also singled out one mid-pregnancy animal which showed no demonstrable brain lesions but which e h b i t e d equally small and distorted AEP’s. One consequence of asphyxia may be a functional disturbance of the auditory system, even when the insult does not lead to pathologic changes detectable with the light microscope. This possibility can only be inferred from the present results, but could be tested experimentally in neurophysiological investigations of unit activity in brain stem auditory nuclei of asphyxiates. In support of this suggestion, Simon (1974) has recently shown that despite the absence of microscopic evidence of neuropathological change, the brains of rats asphyxiated in the newborn period show large increases in catecholamine metabolism, and in particular of norepinephrine turnover.
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The lack of behavioral deficits in performing the auditory discrimination tasks used in this study on the part of the same asphyxiates that showed deviant AEP’s may simply reflect the use of stimulus conditions that were less than optimal in revealing potential behavioral deficits. However, such an explanation would not account for their superiority in acquiring the auditory discrimination. Another possibility, considering the relatively late age at which auditory training commenced, relates to the well-known functional plasticity of the brains of young organisms. Undamaged parts of the brain may assume functions critical to mediation of auditory behavior, although the electrodes recording the AEP over auditory areas may continue to reveal cell loss or neural dysfunction along auditory pathways. In support of this argument are the findings of Faro and Windle (1969) who noted neurological recovery dcspitc progrcssive secondary degeneration in the brains of asphyxiated monkeys.
Notes ‘Other tests administered to these animals included a series of 8 color, form, and pattern discrimination tasks, and 4 delay-type tasks, including 0-, 5 - , and 10-sec delays and delayed alternation. In addition, observations were made of the animals’ individual and group-cage behavior at 18 and 30 months of age. Visual evoked potentials, as well, were measured at repeated intervals. No deficit was found in the asphyxiate groups in any of the cognitive tasks, nor were consistent differences between groups seen in the visual evoked potentials. However, signs of social inadequacy in feeding contests or “bizarre” behavior were seen in the asphyxiates at 18 months of age. The latter behaviors included rocking, stereotypy, self-clasping, and huddling. These were largely dissipated by 30 months of age. Additional details as to the methods and results of these preliminary observations are available from the authors. Dr. Jonathan Wegener (personal communication) has trained 6 juvenile monkeys subjected to 14 min of total asphyxia at birth and 4 comparably treated controls (including cesarean delivery) o n a series of auditory discriminations. The task required a shuttlebox avoidance response to avert shock. The term asphyxiates showed n o clear inferiority compared to the controls. Instead, they learned a frequency discrimination as easily as the controls, 1 pattern discrimination in fewer average trials, a 2nd in more trials, and a 3rd in the same number of trials as the controls. None of these differences was statistically significant. Wegener also noted that the asphyxiates were quieter, more attentive, and less agitated than the controls in the testing situation. This work was supported in part by Grant MH-12568 and by Research Scientist Award K5-14915 (Dr. Mirsky) from NIMH, ADMHA. The assistance of Richard Mandel, Nancy Harman, Laura Mirsky, and Carol V. Mirsky is gratefully acknowledged. Dr. Myers is Chief of the Laboratory of Perinatal Physiology, NINCDS, National Institutes of Health.
References Adamsons, K., Mueller-Heubach, E., and Myers, R. E. (1971). Production of fetal asphyxia in the rhesus monkey by administration of catecholamines t o the mother. Am. J. Obstet. Gynecol., 109:248-262. Brann, A. W., and Myers, R. E. (1975). Central nervous system findings in the newborn monkey following severe in utero partial asphyxia. Neurology, 25:327-338. Berman, D., Karalitzky, A. F., and Berman, A. .I.(1971). Auditory thresholds in monkeys asphyxiated at birth. Exp. Neurol., 31 :140-149. Casseday, J. H., and Neff, W. D. (1975). Auditory localization: Role of auditory pathways in brain stem of the cat. J. Neurophysiol., 38:842-858.
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Dewson, J. H., and Burlingame, A. C . (1974). Auditory discrimination and recall in monkeys. Science, 187:267-268. Faro, M. D., and Windle, W. F. (1969). Transneuronal degeneration in brains of monkeys asphyxiated at birth. Exp. Neurol., 24:38-52. Ginsberg, M. D., and Myers, R. E. (1976). Fetal lesion injury after maternal carbon monoxide intoxication. Neurology, 26:15-23. Hyman, A., Berman, D., and Berman, A. J. (1971). Deficits in unsignaled avoidance behavior in rhesus monkeys asphyxiated at birth. Exp. Neurol., 30:362-366. Hyman, A., Parker, B., Berman, D., and Berman, A. J. (1970). Deficits in delayed response performance in monkeys deprived of oxygen at birth. Exp. Neurol., 38:420-425. Mirksy, A. F., Tecce, J. J., Harman, N., and Oshima, H. (1975). EEG correlates of impaired attention performance under secobarbital and chlorpromazine in the monkey. Psychopharmacologia (Berl.), 41 135-41. Myers, R. E. (1967). Models of asphyxia1 brain damage in the newborn monkey. Paper presented at 2nd Pan American Congress of Neurology. San Juan, Puerto Rico. Myers, R. E. (1969). The clinical and pathological effects of asphyxiation in the fetal rhesus monkey. In K. Adamsons (Ed.), Diagnosis and Treatment of Fetal Disorders. New York: Springer-Verlag. Pp. 226-249. Myers, R. E. (1971a). Brain damage induced by umbilical cord compression at different gestational ages in monkeys. In E. I. Goldsmith and J. Moor-Jankowski (Eds.), Second Conference on Experimental Medicine and Surgery in Primates. New York: S. Karger. Pp. 394-425. Myers, R. E. (1971b). Conditions leading to perinatal brain damage in the non-human primate. In P. G. Crosignani and G. Perdi (Eds.), Fetal Evaluation During Pregnancy and Labor: Experimental and Clinical Aspects. New York: Academic Press. Pp. 175-199. Myers, R. E., Mueller-Heubach, E., and Adamsons, K. (1973). Predictability of the state of fetal organization from a quantitative analysis of the components of late deceleration. Am. J. Obstet. Gynecol., 115:1083-1094. Ranck, J. B., and Windle, W. F. (1959). Brain damage in the monkey, Macaca mulatta, by asphyxia neonatorum. Exp. Neurol., 1:130-154. Sechzer, J. A. (1969). Memory deficits in monkeys brain damaged by asphyxia neonatorum. Exp. Neurol., 24:497-506. Sechzer, J. A., Faro, M. D., and Windle, W. F. (1973). Studies of monkeys asphyxiated at birth: Implications to minimal cerebral dysfunction. Semin. Psychiatr., 5 :19-34. Simon, N. (1974). Long term effects of neonatal asphyxia in the rat. Unpublished Ph.D. dissertation, Department of Biochemistry, Division of Medical and Dental Sciences, Boston University. Windle, W. F., Becker, R. F., and Weil, A. (1944). Alterations in brain structure after asphyxiation at birth. An experimental study in the guinea pig. J. Neuropathol. Exp. Neurol., 3:224-228.
Over 30 years ago, W. F. Windle published the first in a series of papers describing the effects of asphyxia neonatorum in guinea pigs and monkeys (Faro & Windle, 1969; Ranck & Windle, 1959; Windle, Becker, & Weil, 1944). In the monkey, total asphyxia at birth results in neuropathological changes in the brain stem, thalamus, basal ganglia, and cerebellum (Ranck & Windle, 1959). Particularly affected are the inferior colliculi, but damage is also incurred by other sensory relay nuclei of the auditory, somesthetic, and vestibular pathways. Myers (1967) has confirmed these findings and, in addition, shown that vulnerability to the effects of total asphyxia in the monkey is related to
Reprint requests should be sent to Allan F. Mirsky, Laboratory of Neuropsychology, Division of Psychiatry and Department of Neurology, Boston University School of Medicine, 80 E. Concord Street, Boston, Mass. 02118, U.S.A. Received for publication 7 April 1977 Revised for publication 28 April 1978 Developmental Psychobiology, 12(4):369-379 (1979) Q 1979 by John Wiley & Sons, Inc.
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gestational age (Myers, 1971a). Thus, fetuses of 90-95 days can withstand up to 30 min of cord clamp without sustaining detectable pathology, whereas only 10 niin in the term fetus (165 days) is sufficient to inflict serious damage. Myers and his colleagues have also developed alternative methods for inducing pre- arid perinatal asphyxia in monkeys which may model more closely the events and consequences of :I) difficult labor and delivery (Adamsons, Mueller-Heubach, & Myers, 1971 ; Brann 8c Myers, 1975; Myers, 1969, 1971b; Myers, Mueller-Heubach, & Adamsons, 1973). These procedures, involving a prolonged period of partial asphyxia, result in neuropathology of the cortex and basal ganglia. The functional consequences of pre- and perinatal asphyxia, as might be measured behaviorally or electrophysiologically, have received only limited experimental attention. Monkeys subjected to total asphyxia at birth show a wide variety of permanent and transient behavioral deficits on tasks of delayed response, unsignalled avoidance, general reactivity, visual discrimination, visually guided behavior, and loconiotor development (Hyman, Berman, & Berman, 197 1; Hyman, Parker, Berman. 81 Berman, 1970; Sechzer, 1969; Sechzer, Faro, & Windle, 1973). In light of the vulnerability of the auditory system to total asphyxia at birth, it is surprising that only one investigation has been concerned with subsequent auditory behavior. Using an avoiclance paradigm Berman, Karalitzky, and Berman (1971) found an increase in auditory thresholds of monkeys asphyxiated at birth. The present work provides some preliminary behavioral and electrophysiological observations on auditory functioning in monkeys subjected prenatally or at term to total and/or partial asphyxia.
Methods Asphyxiations The asphyxiations were carried out while the fetuses (Mucaca vnulatfa) remained in the uterus. The partial asphyxia of 5 term fetuses was accomplished by constricting tlie maternal aorta (Myers et al., 1973) or by administering carbon monoxide t o the mother (Ginsberg & Myers, 1976). In a single instance, the asphyxia resulted from the procedures (catheterization of the mother) used to prepare the animal for study. In 3 animals the partial asphyxia was followed by a period of total asphyxia (ranging froin 1-15 min) brought about by clamping the umbilical cord. The magnitude of asphyxia was monitored and controlled after catheterization of the fetal femoral artery by measuring blood pH, pO2, and pCOz (Brann & Myers, 1975). In the 3 mid-pregnancy (94-98 day gestational age) asphyxiations, the umbilical cord was exposed through a small incision into the uterus and tightly occluded manually for a period of 30-35 min. This duration was selected on a basis of prior study (Myers, 1971a) to be sufficient i:o produce damage to structures in the brain stem. At the end of this time, the umbilical cord (or the heart) was injected with epinephrine and gentle massage of the heart was instituted through the fetal chest wall to restore circulation and heart beat. The various incisions were closed in anatomical layers and the pregnancy allowed to continue until term.
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Acquisition, Housing and Rearing Conditions The mid-pregnancy animals and 2 of the controls (C-M and C-C) remained with their mothers for 3 weeks after birth. Thereafter, they were separated from the mothers and hand-reared until they were able to feed themselves independently, usually at 2-3 months of age. All of the term animals were hand-reared from the time of birth. Of the remaining control animals, C-L was hand-reared from the age of 1 week whereas C-W, a feral animal, was acquired for the present study at the age of 2 years. All animals were housed in individual cages in the busy and active general monkey housing area. Except for C-W, all were subjected to large amounts of individual handling from an early age.
Experiment I: Auditory Discriminations Subjects The subjects were 5 monkeys asphyxiated at term and 3 unasphyxiated control monkeys. All were 16-18 months of age at the beginning of training except for 1 control (C-W) which was 1 year older.
Testing and Training Procedures During training, the monkeys were water deprived; monkey chow was available ad libitum. Animals were tested while seated in a small restraining chair in a testing chamber (105 cm high x 44.5 cm wide x 53.3 cm deep). The combined manipulanda-discriminanda were a triangular array of switches mounted on the wall facing the animal. Reinforcement was a drop of commercial orange-flavored sugar drink delivered through a tube near the mouth. The animals began training, by successive approximations, on a self-paced auditory discrimination task (Dewson & Burlingame, 1974) in which they were to press for 2 sec the top center switch of the triangular array. The 2-sec hold resulted in an auditory stimulus presented through a center-mounted speaker. A discriminative response involving the left or right switch was then required depending on the auditory stimulus. In 1 version of the task, the monkeys were reinforced for pressing the left switch for white noise or the right switch for a 2500-Hz tone. In the 2nd version, a discrimination between 2 tones (2500 vs 4800 Hz) was required. After an error, the same discriminative stimulus was presented on succeeding trials until a correct response was made. The animals worked for 100 trials at the beginning and 200 trials later in training. Visual cues (i.e., illuminating the “correct” switch) were used during shaping to help establish the auditory discrimination and also later in training as prompts. Eventually the animals reached a criterion level of at least 80% correct responding without visual cueing.
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Results Two term animals (T-Z and T-C) were so severely impaired in motor capacity that they could not be tested for auditory discrimination. The remaining asphyxiates showed no deficits in auditory discrimination learning and, in fact, were superior as a group to the controls in acquisition up to the level of 80% correct responses. Two of the 3 asphyxiates also reached more stringent criterion levels more rapidly than any of the control animals (see Table 1). On the tone-tone discrimination task, none of the animals in either the control or aphyxiated groups achieved levels of correct responding greater than 78% despite extensive training. Group differences were not evident on this task.’ We add here some behavioral observations made in the course of testing thcse animals. The auditory discriminations were difficult for the animals to learn, requiring in some cases up to 8 months of training. The incidence of “troublesome’’ behavior or screaming “fits,” especially when frustrated, was high among control animals. At times: testing had to be suspended temporarily. Only 1 asphyxiated animal displayed such behavior, but to a lesser degree which did not require an interruption of testing.
Experiment 11: Auditory Evoked Potentials (AEP’s) Subjects The subjects were 5 monkeys asphyxiated at term, 3 monkeys asphyxiated in mid-pregnancy, and 3 unasphyxiated controls. One monkey was tested on a single occasion at 6 months of age and the rest were tested several times between the ages o:f 14 and 36 months.
Procedure The animals were tested in a restraining chair fitted with head restraints. The EEG was recorded using a Grass Model 7 polygraph and a Technical Measurements
TABLE I . Noise-Tone Discrimination: Number of Testing Days (200 TrialslDay) to Reach Given Level.
Controls
Term Animals
Animal
70%”
75%a
80%a
85%
90%
C-M
35
41
61
66
70
C-L
35
35
37
57
58
c-w
20
25
28
30
35
T-R
18
19
27
44
47
T-E
16
18
22
28
29
T-S
7
8
11
17
18
aPerformance levels at which no overlap existed between control and term animals.
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Corporation FM magnetic tape recorder for later processing on a LINC 8 computer. The recordings were made with subdermal electrodes inserted into the scalp over the frontal, anterior temporal, and occipital regions bilaterally and over the central vertex. An additional midfrontal electrode served as ground. Auditory stimuli, presented via .03-W, 8-52 earphones affixed to the head restraints, were produced by a GrasonStadler model 901 B noise generator. The generator output was put through a 10-msec time gate, and the signals produced were presented binaurally at 2 intensities (72 and 82 dB as estimated with a General Radio sound level meter) and at 2 rates (l/sec and 1/2 sec). The recordings (300 stimuli in each condition) were made at full room illumination. The animals invariably remained fully alert throughout the recording period. The AEP analysis was performed on the vertex to the left temporal and vertex to the right temporal leads. Although recordings were made at a number of ages, only those from around 25 months of age (nearest the completion of the auditory discrimination testing) are presented.
Results The AEP's of asphyxiates differed from those of controls (see Fig. 1) along 1 or more of the following dimensions: (a) morphology; (b) latency and side differences; (c) amplitude. (a) Morphology: The AEP's of asphyxiated animals often lacked the pronounced P2-N3 waves apparent in the responses of control animals. Some of the asphyxiates (T-S and M-E in Fig. 1) showed an additional negative wave following the N2 complex that was rarely present in control animals. Pooling the 2 intensities of click and 2 rates of click presentation showed this component to be present significantly more often in asphyxiates than in controls (X' test, p < .001). (b) Latency and side differences: In control animals, component N1 peaked at 15-25 msec after the click. Differences in the latency of this component recorded from the left vs right side of the head of a given animal never exceeded 5 msec. The latency of this component was either not affected or increased slightly with an increase in stimulus intensity from 72 to 82 dB. In the term asphyxiates, the latency range for this component was 10-30 msec with the shortest and longest latencies attributable to animals with inferior colliculus damage. These monkeys differed most from the controls at the lower intensity (72 dB) and slower presentation rate (1/2 sec). One of the animals with inferior colliculus damage showed differences in the latency of the N1 components on the left vs right side of the head as great as 20 msec. In the mid-pregnancy group, the latency of N1 ranged from 10 to 35 msec, both extremes occurring at the higher click intensity. Side differences were considerable, though not always consistent, with 10 to 15 msec discrepancies for each of the mid-pregnancy animals under one or more stimulus conditions. Whereas the latency of N1 was either increased or unaffected by stimulus intensity for controls, 4 of the 7 asphyxiates, including 1 animal with inferior colliculus damage, showed a decrease in N1 latency with an increase in stimulus intensity. Component P1 was the 1st positive component which, in control animals, peaked at 40 to 50 msec. Side differences did not exceed 5 msec. Among the 3 controls, the
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AU DlTORY EVOKED POTENTIALS
ASPHYXIATES
*T-C
INFERIOR COLLlCULUS
Fig. 1. Auditory evoked potentials (AEP’s) to an 8 2 d B 1/2-sec click sourccs for all animals in the study. Also included are AEP’s from T-C, a term asphyxiate with extensive brain damagt:, including bilateral destruction of the inferior colliculi. The total sweep time depicted is 425 msec; in each case the wave form represents an average of 200-300 samples. The latcncies in msec scen in normal animals for the several pcaks shown at thc top of the figure are as follows: N 1 : 15-25; P I : 40-50; N2a: 60-75; N2b: 85-320. Note the relatively flat AEP’s for the 3 animals with inferior colliculus Icsions: T-R, T-Z, and T C and for M-A; and the generally reduced amplitudes of all asphyxiates. The text describes other differences among the groups.
latency of the components was not uniformly affected by either rate or intcnsity changes. The term asphyxiates with demonstrable brain damage (T-S, T-K, T-Z: T-C), when compared to the control animals, tended t o show a slightly slower (not exceeding 5 msec) P1 component at the lower stiinulus intensity (72 dB) and faster rate of presentation ( l / l sec). Under other stimulus conditions the P1 latencies of these animals were comparable to the controls. One mid-pregnancy asphyxiate, M-A, showed a substantial side difference in addition to a grossly deviant P1 latency, with a range of 15-25 msec on the right side and 35-45 msec on the left. The pattern of deviation was also apparent for this animal when considering the N2a component. (c) Amplitude: By far the most striking feature of the AEP’s of asphyxiates was their small amplitude compared to control animals. This is apparent in Figure 1, showing the response to 82-dB clicks presented at 1/2 sec, but was also a feature (of responses at lower click intensities and faster rates.
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The amplitude between P1 and the large peak of the N2 complex completely separates all asphyxiates from the three controls. The lowest amplitudes for each of the AEP components were consistently attributable to the term animals with inferior colliculus damage and to mid-pregnancy asphyxiate, M-A. This animal also showed grossly deviant latencies. In many instances the highest amplitude shown by an asphyxiate did not exceed or, in some cases, even come close to, the lowest amplitude shown by a control. The possibility that the extremely low amplitude AEP’s on the part of some of the asphyxiates (T-Z, T-R, and M-A) was secondary to or correlated with low amplitude, high frequency EEG was tested in these 3 cases by a computerized frequency and amplitude analysis (Mirsky, Tecce, Harman, & Oshima, 1975) of the resting EEG recorded prior to EP testing using the same electrode ptacements as in the AEP analysis. The results indicated that the asphyxiates generating the extremely small AEP’s did not show more low amplitude fast activity than controls. The small amplitude AEP’s on the part of these 3 asphyxiates (T-R, T-Z, M-A) were specific to the auditory modality and not due to the procedural flaws or a general dysfunction which might have affected equally EP’s recorded in all modalities. Two of the 3 controls and 2 of the 5 asphyxiates showing small AEP’s were tested during the same recording session for visual evoked potentials (VEP’s) to photic stimulation, These data indicated that the VEP’s of the asphyxiates were comparable to or even slightly larger than the VEP’s of the controls. Consequently, the degradation of the AEP appears to be a modality-specific effect. Tests at other ages gave the same AEP separation among animals as that presented here.
Histological Examination of Brains The brains of all term and mid-pregnancy asphyxiates were examined grossly and microscopically. No abnormality was seen in the brains of any of the mid-pregnancy animals or of term animal T-E. Definite pathologic changes were seen in the brains of the remaining 4 term animals. Animal T-S exhibited enlargement of the occipital horn of the right lateral ventricle with thinning of the overlying occipital cortex. Animal T-R showed marked nerve cell loss and gliosis in multiple thalamic nuclei including the lateral part of the posterior ventral, and the intralaminar and the lateral parts of the dorsomedial nuclei. In addition, T-R exhibited marked gliotic scars in the inferior colliculus (Fig. 2) and both parts of the superior olivary nuclei. (The normal-appearing inferior colliculus of M-A is shown in Fig. 2, for purposes of comparison with T-R.) The most marked damage was seen in T-Z and T-C. T-Z’s brain showed symmetrical atrophic sclerosis of the postcentral gyri (arm and leg areas), sclerosis and major tissue loss of the putamen and portions of the caudate nucleus and globus pallidus, and scarring of the posterior ventral and centromedial nuclei of the thalamus bilaterally. The inferior colliculi also showed great reduction in size and central scarring. Animal T-C showed a true microcephaly with a major reduction of brain size. Neurological examination of these animals at the time of sacrifice revealed the following abnormalities: Animal T-C was in a permanent state of opisthotonus and required complete hand care. Animal T-Z showed great clumsiness in all actions (more so in the arms than legs) with a scissors gait and increased adductor tone. Its placing and hopping reactions were greatly depressed although both the hands and feet showed grasping reactions. It gave some suggestion of intention tremor and past-pointing.
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(b) Fig. 2. (a) Microphotograph of the brain stem of the term animal T-R showing gliosis and absence of cells in the inferior colliculus; (b) microphotograph of the brain stem of the mid-pregnancy asphyxiate M-A showing apparently normal inferior colliculus.
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Animal T-S exhibited an abnormal posture of the hind legs in sitting, standing and walking. It was observed to walk on his heels, rather than on his toes. It showed some suggestion of a broad base in the hands. Animal T-R manifested some abnormal placement of its hind limbs, and a suggestion of this in the forelimbs. The other animals showed no neurological abnormalities.
Discussion The present investigation was undertaken as a pilot study and, as such, suffers several limitations. The combination of partial and total asphyxia of the term fetuses did not allow us to separate the functional consequences of the 2 procedures. The auditory discrimination tasks were apparently not entirely suited for our monkey subjects since all animals required intensive and extensive training. Finally, only a few animals in each condition were studied. Nevertheless, the results have raised some interesting questions concerning the relationship among behavioral, electrophysiological and neuroanatomical variables in this area of research. Although previous investigations have demonstrated a multiplicity of behavioral impairments on the part of asphyxiated monkeys, we failed to find any evidence of deficit in auditory discrimination in our asphyxiated animals. In fact, the term group, up to the late stages of acquisition, learned the noise-tone discrimination more rapidly than control animals. However, all animals had considerable difficulty in acquiring this task (i.e., up to 8 months of training). Thus, the procedure used might be more a measure of tolerance of frustration/failure than a measure of auditory discrimination ability.2 Two of the control animals were often difficult to test, especially after repeated failure to achieve mastery. This intolerance was less often observed with the asphyxiated animals and suggests the possibility of motivational basis for their superiority. The electrophysiological findings of pathological evoked potentials in the auditory but not the visual modality in the asphyxiates suggest several possibilities. All asphyxiates, regardless of whether damage to the auditory system could be demonstrated, showed AEP’s of overall smaller amplitude and lacking those late components which were often prominent in normals. Many asphyxiates also showed deviant latencies and grossly asynchronous responses from the 2 sides of the head. The amplitude measures of individual AEP components clearly separated animals with inferior colliculus damage from other animals. Casseday and Neff (1975) have reported a similar finding in adult cats with surgical lesions of the inferior colliculus. However, the same measures also singled out one mid-pregnancy animal which showed no demonstrable brain lesions but which e h b i t e d equally small and distorted AEP’s. One consequence of asphyxia may be a functional disturbance of the auditory system, even when the insult does not lead to pathologic changes detectable with the light microscope. This possibility can only be inferred from the present results, but could be tested experimentally in neurophysiological investigations of unit activity in brain stem auditory nuclei of asphyxiates. In support of this suggestion, Simon (1974) has recently shown that despite the absence of microscopic evidence of neuropathological change, the brains of rats asphyxiated in the newborn period show large increases in catecholamine metabolism, and in particular of norepinephrine turnover.
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The lack of behavioral deficits in performing the auditory discrimination tasks used in this study on the part of the same asphyxiates that showed deviant AEP’s may simply reflect the use of stimulus conditions that were less than optimal in revealing potential behavioral deficits. However, such an explanation would not account for their superiority in acquiring the auditory discrimination. Another possibility, considering the relatively late age at which auditory training commenced, relates to the well-known functional plasticity of the brains of young organisms. Undamaged parts of the brain may assume functions critical to mediation of auditory behavior, although the electrodes recording the AEP over auditory areas may continue to reveal cell loss or neural dysfunction along auditory pathways. In support of this argument are the findings of Faro and Windle (1969) who noted neurological recovery dcspitc progrcssive secondary degeneration in the brains of asphyxiated monkeys.
Notes ‘Other tests administered to these animals included a series of 8 color, form, and pattern discrimination tasks, and 4 delay-type tasks, including 0-, 5 - , and 10-sec delays and delayed alternation. In addition, observations were made of the animals’ individual and group-cage behavior at 18 and 30 months of age. Visual evoked potentials, as well, were measured at repeated intervals. No deficit was found in the asphyxiate groups in any of the cognitive tasks, nor were consistent differences between groups seen in the visual evoked potentials. However, signs of social inadequacy in feeding contests or “bizarre” behavior were seen in the asphyxiates at 18 months of age. The latter behaviors included rocking, stereotypy, self-clasping, and huddling. These were largely dissipated by 30 months of age. Additional details as to the methods and results of these preliminary observations are available from the authors. Dr. Jonathan Wegener (personal communication) has trained 6 juvenile monkeys subjected to 14 min of total asphyxia at birth and 4 comparably treated controls (including cesarean delivery) o n a series of auditory discriminations. The task required a shuttlebox avoidance response to avert shock. The term asphyxiates showed n o clear inferiority compared to the controls. Instead, they learned a frequency discrimination as easily as the controls, 1 pattern discrimination in fewer average trials, a 2nd in more trials, and a 3rd in the same number of trials as the controls. None of these differences was statistically significant. Wegener also noted that the asphyxiates were quieter, more attentive, and less agitated than the controls in the testing situation. This work was supported in part by Grant MH-12568 and by Research Scientist Award K5-14915 (Dr. Mirsky) from NIMH, ADMHA. The assistance of Richard Mandel, Nancy Harman, Laura Mirsky, and Carol V. Mirsky is gratefully acknowledged. Dr. Myers is Chief of the Laboratory of Perinatal Physiology, NINCDS, National Institutes of Health.
References Adamsons, K., Mueller-Heubach, E., and Myers, R. E. (1971). Production of fetal asphyxia in the rhesus monkey by administration of catecholamines t o the mother. Am. J. Obstet. Gynecol., 109:248-262. Brann, A. W., and Myers, R. E. (1975). Central nervous system findings in the newborn monkey following severe in utero partial asphyxia. Neurology, 25:327-338. Berman, D., Karalitzky, A. F., and Berman, A. .I.(1971). Auditory thresholds in monkeys asphyxiated at birth. Exp. Neurol., 31 :140-149. Casseday, J. H., and Neff, W. D. (1975). Auditory localization: Role of auditory pathways in brain stem of the cat. J. Neurophysiol., 38:842-858.
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Dewson, J. H., and Burlingame, A. C . (1974). Auditory discrimination and recall in monkeys. Science, 187:267-268. Faro, M. D., and Windle, W. F. (1969). Transneuronal degeneration in brains of monkeys asphyxiated at birth. Exp. Neurol., 24:38-52. Ginsberg, M. D., and Myers, R. E. (1976). Fetal lesion injury after maternal carbon monoxide intoxication. Neurology, 26:15-23. Hyman, A., Berman, D., and Berman, A. J. (1971). Deficits in unsignaled avoidance behavior in rhesus monkeys asphyxiated at birth. Exp. Neurol., 30:362-366. Hyman, A., Parker, B., Berman, D., and Berman, A. J. (1970). Deficits in delayed response performance in monkeys deprived of oxygen at birth. Exp. Neurol., 38:420-425. Mirksy, A. F., Tecce, J. J., Harman, N., and Oshima, H. (1975). EEG correlates of impaired attention performance under secobarbital and chlorpromazine in the monkey. Psychopharmacologia (Berl.), 41 135-41. Myers, R. E. (1967). Models of asphyxia1 brain damage in the newborn monkey. Paper presented at 2nd Pan American Congress of Neurology. San Juan, Puerto Rico. Myers, R. E. (1969). The clinical and pathological effects of asphyxiation in the fetal rhesus monkey. In K. Adamsons (Ed.), Diagnosis and Treatment of Fetal Disorders. New York: Springer-Verlag. Pp. 226-249. Myers, R. E. (1971a). Brain damage induced by umbilical cord compression at different gestational ages in monkeys. In E. I. Goldsmith and J. Moor-Jankowski (Eds.), Second Conference on Experimental Medicine and Surgery in Primates. New York: S. Karger. Pp. 394-425. Myers, R. E. (1971b). Conditions leading to perinatal brain damage in the non-human primate. In P. G. Crosignani and G. Perdi (Eds.), Fetal Evaluation During Pregnancy and Labor: Experimental and Clinical Aspects. New York: Academic Press. Pp. 175-199. Myers, R. E., Mueller-Heubach, E., and Adamsons, K. (1973). Predictability of the state of fetal organization from a quantitative analysis of the components of late deceleration. Am. J. Obstet. Gynecol., 115:1083-1094. Ranck, J. B., and Windle, W. F. (1959). Brain damage in the monkey, Macaca mulatta, by asphyxia neonatorum. Exp. Neurol., 1:130-154. Sechzer, J. A. (1969). Memory deficits in monkeys brain damaged by asphyxia neonatorum. Exp. Neurol., 24:497-506. Sechzer, J. A., Faro, M. D., and Windle, W. F. (1973). Studies of monkeys asphyxiated at birth: Implications to minimal cerebral dysfunction. Semin. Psychiatr., 5 :19-34. Simon, N. (1974). Long term effects of neonatal asphyxia in the rat. Unpublished Ph.D. dissertation, Department of Biochemistry, Division of Medical and Dental Sciences, Boston University. Windle, W. F., Becker, R. F., and Weil, A. (1944). Alterations in brain structure after asphyxiation at birth. An experimental study in the guinea pig. J. Neuropathol. Exp. Neurol., 3:224-228.
