NEONATAL ASPHYXIA IN THE RAT: GREATER VULNERABILITY OF MALES AND PERSISTENT EFFECTS ON BRAIN MONOAMINE SYNTHESIS

Journoi OJ Neerochrmisrry. 1976. Vol. 26. pp. 893-900. Pergamon Press. Printed in Great Britain.

NEONATAL ASPHYXIA IN THE RAT: GREATER VULNERABILITY OF MALES AND PERSISTENT EFFECTS ON BRAIN MONOAMINE SYNTHESIS

NICOLE SIMON' and L. VOLICER Department of Pharmacology and Experimental Therapeutics, Boston University School of Medicine,

Boston, MA 02118, U.S.A.

(Received 17 June 1915. Accepted 10 October 1975)

Abstract-In the rat, neonatal asphyxia produced by suffocation did not leave permanent visible lesions in thc brain, nor did it result in permanent motor impairment, although a delay in the development of some reflexes was observed. A transient retardation of body and brain growth, which was more pronounced in males, was found. By 5-6 weeks of age, body and brain weights of asphyxiated rats were no longer significantly different from control animals. However, an increase in brain norepinephrine synthesis was found to persist after maturation. An alteration of serotonin metabolism was found after maturation only in asphyxiated males. The possibility that neonatal asphyxia in the rat is a model for abnormal development of monoamine metabolism, relevant to early childhood behavior disorders such as infantile autism or the syndrome of minimal brain dysfunction, is discussed.

RESEARCH on the effects of asphyxia in the perinatal period has revealed that in several species, including man, the resulting injury to the brain may be confined to subcortical structures without involvement of the cerebral hemispheres (CLARK & ANDERSON, 1961;

et a/., 1964; CHEN et a/., 1965; BRAND & BIGNAMI, 1969; WINDLE, 1969a; TOWBIN, 1970; MYERS, 1972; NORMAN, 1972; GRIFFITHS & LAURENCE, 1974; GRUN- NET et al., 1974). Research on monamines in the central nervous system indicates that their normal metabolism depends upon integrity of some of the brainstem areas that are most vulnerable to perinatal asphyxia (JOUVET, 1969; POIRIER et al., 1969; HARVEY & GAL, 1974). We chose to study neonatal asphyxia in the rat as a possible naturalistic model for abnormal development of central monoaminergic neural networks.

It is now well established that, in the monkey, two entirely different patterns of brain damage result from oxygen deprivation around the time of birth, depend- ing upon whether the insult is a brief acute episode of total asphyxia or a prolonged partial disruption of the oxygen supply (MYERS, 1972). Ten-15 min of total asphyxia produced by umbilical cord clamp before the neonate is allowed to breathe, results in a highly predictable pattern of small, focal lesions, most prominent in brainstem auditory structures, reti- cular formation and the thalamus. The vulnerability of these subcortical sites follow a rank order predicted

Predoctoral Trainee of the National Institute of Men- tal Health.

Present address: Behavioral Sciences Division, Bolt Beranck and Newman, Inc., 50 Moulton Street, Cam- bridge, MA 02138, U.S.A.

ROZDILSKY & OLSZEWSKI, 1961; CHEN, 1964; JILEK

by studies of differential circulatory rate in the brain (KETY, 1962) and order of myelin development (YAK- OVLEV & LECOURS, 1967). Thc focal lesions result from rapid production of carbon dioxide and acidosis in these areas of high metabolic activity (WINDLE, 1966; CHEN et al., 1971). Animals with this pattern of focal brainstem lesions do not become spastic or suffer permanent impairment of motor functions (WINDLE, 1969~). The effects of a prolonged partial deficiency of oxygen (hypoxia) are entirely different. The basal ganglia and paracentral cortex are often the most severely damaged after prolonged hypoxia. The involvement of these two motor systems is the charac- teristic pattern underlying the affliction of spastic ccr- ebral palsy and prolonged partial asphyxia results in clear neurological signs.

Early childhood behavior disorders such as infantile autism are not neccessarily accompanied by any hard neurological signs (motor manifestations): hence the focal lesions of brainstem centers seen after a brief episode of total asphyxia at birth may be a more likely cause for this type of disorder. SIMON (1975) has discussed the possible involvement of brainstem auditory nuclei in the language disorder of autistic children. WINDLE (1963, 19696); SECHZER et nl. (1973) believe that the focal lesions of the brainstem and thalamus seen after an acute asphyxic insult may un- derly the clinical syndrome of Minimal Brain Dys- function (MBD). Perinatal injuries have been found to be an important etiological factor underlying both autism and MBD (SECHZER et al., 1973; LOBASCHER ef al., 1970; CHESS, 1971) and disorders of monoamine metabolism have been implicated in these behavioral disorders of childhood (COLEMAN, 1973 ; WENDER, 1972).

893

894 NICOLE SIMON and L. VOLICER

Metabolic anomalies in children are generally thought t o be hereditary. O u r study was designed to investigate whether long term biochemical dysfunc- tions could result from an exogenous insult such as perinatal asphyxia. We chose the rat for our studies because, in the neonatal rat, acute or chronic anoxia affects only brainstem areas and the injury appears to be transient (JILEK et ul., 1964). With maturation, however, there is a residual defect in acquisition and cxtinction of conditioned reflexes, which JILEK et al. (1964) suggested may be due to a long-term alteration of sub-microscopic structure or metabolism in the nervous system.

MATERIALS AND METHODS Materials. Radioactive-~-[3,5-'H]tyrosine (30-50 Ci/

mmol) and ~-[~H]tryptophan, generally labelled (1-5 Ci/ mmol) were purchased from New England Nuclear COT. (Boston). Ion-exchange resin (AG 50-X4, 2 W 0 0 mesh) was purchased from Biorad Labs (Richmond, CA). Alu- minum oxide (alumina) was purchased from the British Drug Houses, LTD. (obtained through Gallard-Schlesinger Chemical Mfg. Corp., Carle Place, NY). Amines and amino acids used for standards were purchased from Calbiochem (LaJolla, CA), Nutritional Biochemicals (Cleveland, OH) and Sigma Chemical Co. (St. Louis, MO). Tris, hydroxy- methyl amino methane, buffer was purchascd from Sigma Chemical Co. (St. Louis). Otherwise, analytical grade re- agents purchased from Fisher Scientific Co. (Fair Lawn, NJ) were used throughout all procedures. Pregnant rats were obtained from the Charles River Breeding Labs. (Wilmington, MA).

Animal treatment. The pregnant rats were shipped on the fourteenth to sixteenth day of gestation and placed, upon arrival, in individual breeding cages. Within 24 h after a natural birth, the mother was removed from the nest cage. The infant rats were weighed and individually marked by cutting off one digit of either the left or right forepaw; this was described by SMART & DOBBING (1971~) as a non-traumatic procedure. Littermate pairs were then selected according to sex and birthweight for long-term comparisons. One member of each pair was subjected to asphyxia and the other kept as a control. We sometimes transferred a pair from one litter to another to equalize litter size; in general, 6 - 8 rats were reared in each cage.

The control littermates were placed in the corners of the nest cage during the asphyxiation of the experimental pups; this kept the pups isolated from each other, but the control animals were not subjected to any additional stress. The experimental animals were asphyxiated in 12 cm' capacity. air-tight vials. The duration of asphyxia was between 45 min and 2 h at room temperature (24-28" C). Animals were removed from the vials when gasping could no longer be elicited by tapping the side of the vial, the mouth had closed and the pup appeared pale and flaccid. The animals were placed, supine, on a paper towel cover- ing a heating pad, which maintained a temperature of 34°C and thcy were dried off with absorbant tissue. Resusci- tation was accomplished by intermittently massaging or softly tapping the chest and applying, at intervals, a slow stream of air to the nose and mouth.

The mother was returned to the nest cage only after the resuscitated pups had been replaced. There was never

any maternal rejection of the asphyxiated pups. All animals were weaned between 21 and 24 days of age.

Experimental procedures. Reflex development was tested in the pre-weanling pups according to the schedule of SMART & DOBBING (1971~). A few rats were sacrificed 20 or 40 days after the asphyxic insult and their brains k e d and stained according to the method of NAUTA (1957). Most of the animals were allowed to mature and were used for biochemical studies between 5 and 6 weeks of age.

At the time of sacrifice, the maximum age difference between litters was 4 days. Six-nine litters were used lor each study of monoamine turnover and paired experimental-control littermates were selected from each litter for every time-point measurement.

Radioactive L-tyrosine or L-tryptophan was injected into the tail vein. The amount injected was about 100 pCi/rat, contained in 0.5 ml of saline/100 g wt of the animal. Each animal was killed by decapitation exactly 90, 120 or 150min after injection. The brain was quickly removcd, blotted and the lower brainstem trimmed off by a cut directly behind the cerebellum; the olfactory lobes were trimmed off by a cut directly in front of the frontal lobes. The brain was wrapped in aluminum foil and immediately frozen on dry ice. The frozen brains were kept for up to 1 week in a freezer at -4°C.

The brains were homogenized in 4 vol of ice cold 0.4 M-perchloric acid containing 0.050/, sodium metabisul- file. Monoamines in the supernatants of the brain homo- genates were separated from their precursor amino acids by ion exchange columns as described by NEFF et al. (1971), with some modifications. Norepinephrine was eluted with 1 . 0 ~ rather than O~M-HCI . Better recovery of serotonin was achieved by elution with saturated trisodium phos- phate than with the 0 . 5 ~ solution used by NE=F et a/. (1971). The norepinephrine and dopamine eluates were further purified on washed alumina according to the method of CHANG (1964). Both catecholamines were measured fluorometrically in the final effluent from alumina by the method of LAWRTY & TAYLOR (1968).

Calculations. The mean efficiency in counting of radioactivity was found to be the same (30%) for all of the final aqueous eluates, hence this factor was not carried through the calculations. To compute the specific activity (SA) for each amine and amino acid, the counts per minute (c.p.m.) were multiplied by the molecular weight (MW x 10 3,

and three factors to correct for the fraction of the total tissue sample used for counts (F), the recovery (R), and the fraction of tritium ions remaining after conversion to amine (T):

c.p.m. x MW x 1 1 1 L F R T

x - x - x - - . SA =

For serotonin counts, corrections were made for the leakage of free tritium released from the generally labelled tryptophan during the separation procedure. This leakage was estimated by carrying standards of the tritium labelled tryptophan through all the steps of the procedures for iso- lation of serotonin and the fraction (f) of tritium counts in the serotonin eluate was measured. Then, for serotonin, c.p.m. = (c.p.m.5_HI - f x c.p.m.Tr,p). NEFF et al. (1971) estimated that of the twelve hydrogen atoms in the tryptophan molecule, two would be lost in conversion to serotonin and thus 1/6 of the 'tritium radioactivity should be assumed to be lost. We therefore set T = 5/6 for serotonin.

Neonatal asphyxia in the rat 895

It was assumed that 1/2 of the tritium ions would be dis- placed during hydroxylation of tyrosine, which was labelled in the 3,5 positions of the phenyl ring. We therefore set T = 1/2 for dopamine and norepinephrine.

The logarithm of the specific activities, for each animal, were used to compute linear regression curves with respect to time. Values of specific activity for calculation of the synthesis rate constant wcrc calculated from the slope and intercept of the regression lines. at time points 90, 100, 110, 120, 130, 140 and 150. The synthesis rate constant (K) was computed for five 20-min intervals, using the for- mula derived by NEFF et al. (1971):

dMjdi K = - A - M

where dM/dt is the rate of decline of monoamine activity computed between time points tl and t 2 as:

Mf2 - M,, dM/dt = t 2 - t l

and A - M is the average difference between amino acid activity and monoamine activity in the interval, computed by the approximation:

(A - MI,> + (A - M)t2 A - M = 2

RESULTS Survival and vulnerability to asphyxia

Only about half of the experimental animals sur- vived a severe asphyxic insult. Mean death rate in eight experiments (n = 186) was 49 & 7% for males and 47 8% for females. The survivors appeared to be quite depressed during the first 24 h after asphyxia. Most did not gain a normal amount of weight during this period and many lost up to 0.5 g in the first 24 h after asphyxia. Those animals with large weight loss (0.5-1 g) or those who appeared jaundiced (with yel- low discoloration of the skin), often died within the first 24-48 h after asphyxiation.

Control animals (n = 145) gained 1.03 & 0.06g in the first 24 h after birth. Only one control animal lost weight in the first 24 h, a female pup who lost 0-75 g. In contrast to this the average weight gain of 122 experimental animals 24h after asphyxia was only 0.21 +_ 0.06g; this included 37 animals (30.3%) who lost weight during the first 24 h.

It was thought that vulnerability to asphyxia might be related to the metabolic rcquirements of the animal and that this might be related to birth weight and sex. Male animals were significantly heavier at birth (7.21 & 0.06g n = 90) than females (667 k 0.07 g, n = 91 j and 35.9% of the males, as opposed to 24.6% of the females, lost weight during the first 24 h after asphyxia. Computation of correlation coefficient for initial weight loss/gain with respect to birthweight however was not significant for either males (r = -0-12,n = 82) or females (r = 0.02, n = 57). There was a significant correlation of initial weight gain/loss with respect to duration of asphyxia, at room temperature (24-28"C), for female pups (P = -056, n = 46, P < 0001); this was not significant for male pups ( r = -0.16, n = 55).

L : .

0 2 4 6 8 1 0 1 2 d a y s

FIG. 1. Weight gain in normal males (0-0) , n = 8, normal females (+-.), n = 19, asphyxiated males (o----o), n = 10 and asphyxiated females (@---a),

n = 18.

Short-term growth retardation Growth retardation persisted in the first 2 weeks;

this is illustrated in Fig. 1, for the data from one experiment. This pattern of early growth retardation was strikingly similar in all experiments. A three-way analysis of variance of growth rate indicates that the differences between asphyxiated and control animals are highly significant (P < 0.001), while those between males and females are not; the effect of asphyxiation is however significantly greater for males than females in retarding growth ( P < 0.001). The weight differences for paired male and paired female littermates from another experiment are shown in Fig. 2; these differences are significantly larger in males (P < 0.0016).

Brain growth was also retarded in asphyxiated animals. The average brain weight (& s.E.M.) of 25 male pups 1 week after asphyxia was 721.5 i 10.2mg as compared with an average 755.9 & 8.5 mg for 27 control males. In asphyxialed females, the brain weights averaged 703.1 f 9.3 mg (n = 30) in contrast to 725.2 k 8.0mg (n = 28) in control females. A 2-way

t i m e , days

FIG. 2. Weight differences between control and asphyxiated male (+a) and female (0---0) pairs during the first week aftcr asphyxia. Pairs were selccted a priori on the basis of similar birth weight. Numbers beside each point represent the number of pairs weighed on each day.

896 NICOLE SMON and L. VOLICER

analysis of variance on data from three experiments with 7-day old rats indicated that brain weight was significantly lower (P < 0.01) 1 week after neonatal asphyxia, that the female brain was significantly smaller than the male brain at this age (P < @01), but that brain growth was affected to the same degree in both sexes by neonatal asphyxia. The ratio of brain/body wt was 00378 f 0-008 and 00385 f 0.007 for 7-day old control males and females respectively; this ratio was 0.0413 f 0008 for both asphyxiated males and females; this difference was significant for both males (P < 0-001) and females (P < 0.01). At 5-6 weeks of age the trend for lower brain and body weights was still evident in male animals, but the differences with respect to the control groups were no longer statistically significant.

Reflex development and histology The asphyxiated animals did not become spastic

or noticeably incapacitated in any way. To determine if there were any developmental reflex or motor defi- ciencies, a total of nine experimental and nine control litters, in three separate experiments, were examined according to the method of SMT & DOBBING (1971a). The differenax between control and asphyxiated animals were significant (P < 0.05) in two .of these tests (grasp and negative geotaxis).

The forelimb grasp reflex usually develops between the second and third day after birth (Fox, 1965) and wanes between the sixth and seventh days of life. After neonatal asphyxia, the development of the forelimb grasp was delayed by a full day in male pups; the delay was somewhat smaller in females but an analysis of variance indicated that the males were not significantly more delayed than females.

By the fourth or fifth day of life, the rat pup placed head-down on a 20-degree inclined plane, instinc- tively turns to a head-up position; this is called negative geotaxis. Partial turning (to a horizontal position) begins around the third day of life. In asphyxiated male pups the turning was again delayed by a full day; the female pups were somewhat less delayed, but the male-female difference was not significant.

A histological study, using NAUTA'S (1957) procedure for degenerating fibers, 3 and 6 weeks after asphyxia revealed no focal lesions or visible disruption of neural pathways.

Tryptophan to serotonin metabolism The decline of specific activity of tryptophan and

serotonin id the brain, 9@150 min after injection of tritium-labelled tryptophan into the tail-vein of 5-6-week old rats is shown in Fig. 3 for males and Fig. 4 for females. In male rats, the specific activity of tryptophan was slightly higher at all time points and steeper decline in the specific activity of serotonin was found in asphyxiated males compared with the male control group (Fig. 3); and the computed rate

1 0 *' 90 120 I50

FIG. 3. Decline in specific activity of tritium-labelled tryptophan (0) and serotonin (0) in male rats. The points are mean values and the bars indicate S.E.M. The lines are regression curves computed for control (solid lines) and

asphyxiated (broken lines) animals.

minuter

constant for serotonin was significantly increased (Table 1).

Although the rate of decline in tryptophan activity was steeper in the asphyxiated females, and the specific activity in the serotonin fraction was somewhat higher at all time points (Fig. 4), the rate of decline in serotonin activity was the same in control and asphyxiated animals. There was no significant difference between asphyxiated and control females in the computed rate constant, or in serotonin synthesis rate in nmol/h per g of tissue. The serotonin synthesis rate in the female brain was higher than in the male brain, control or asphyxiated, (Table 1).

Tyrosine and catecholamine metabolism

The decline in specific activity of tyrosine, dopamine and norepinephrine in the brain, 9CL150 min after injection of tritium labelled tyrosine into the tail- vein, is shown in Fig. 5. Tyrosine activity was higher at all three time points in asphyxiated animals and

1 ,$, 0 90 120 150

minuter

FIG. 4. Decline in specific activity of tritium-labelled tryptophan (0) and serotonin @) in female rats. The points are mean values and the bars indicate S.E.M. The lines are regression curves computed for control (solid lines) and

asphyxiated (broken lines) animals.


I // 0 " SB rio rbo

minuter

FIG. 5. Decline in specific activity of tritium-labelled tyrosine (U), dopamine (0), and norepinephrine (A). The points are mean values and the bars indicate S.E.M. The lines are regression curves computed for control (solid lines) and

asphyxiated (broken lines) groups of animals.

its decline was somewhat steeper. Norepinephrine activity was also higher at all time points, with a steeper decline during the 9G-150 min interval after injection of its precursor amino acid. Synthesis rate for norepinephrine computed from this data indicates that the rate of norepinephrine synthesis in the asphyxiated animals was nearly double that of t6e control group (Table 1). There was no difference in the decline of dopamine activity, as seen in Fig. 5, and the rate constant for dopamine synthesis was not significantly different in asphyxiated compared with control animals (Table 1). There were no male-female differences evident in the catecholamine data.

DISCUSSION

Growth and development Newborn rats subjected to asphyxia by suffocation

did not become spastic or display any signs of sub-

sequent gross motor impairment. Only with careful, day by day assessement was a delay in the acquisition of some motor responses revealed. Permanent necro- tic lesions were not seen in the brains of asphyxiated animals. The most striking result of neonatal asphyxia in the rat was growth retardation, with asphyxiated male animals showing a greater lag in weight-gain than asphyxiated females. Brain growth was also retarded in the asphyxiated animals, but to a lesser degree than body growth. By 5-6 weeks of age, the body and brain weight differences of the asphyxiated and control animals were no longer statistically significant.

These results stand in contrast to the studies of perinatal malnutrition (SHOEMAKER & WUKTMAN, 1971; ShlART & DOBBING, 1971a), in which weight differences between nutritionally deprived and control rats did not become significant until 5 days after birth (SMART & DOBBMG, 1971b), but the degree of growth retardation increased with maturation. ' I le effects on the rat of asphyxia and malnutrition in the perinatal period then can be clearly differentiated; they are not equivalent insults. Growth retardation was also observed by FRANCESCONI & MAGER (1969) in infant rats reared under hypoxic conditions, with normal growth rate seen after return to normal atmospheric environment. Prolonged hypoxia and brief total asphyxia thus appear to be related, with rcspect to growth impairment, in the rat.

Measurement of monoamine synthesis

A completely reliable method for the true rate of neurotransmitter formation has still to be devel- oped (MOKOT-GAUDRY et al., 1974). We chose the method of NEFF et al., (1971) because we felt that it was suitable for the measurement of differences between our control and asphyxiated groups. We found no indication of severe alteration of neuronal development after neonatal asphyxia in the rat that would make the asphyxiated animals incomparable

TA~LE 1. EFFECT OF NEONATAL ASPHYXIA ON SYNTHESIS RATE OF BRAIN MONOAMINES IN &DAY OLD RATS

(C = Control, A = Asphyxiated Within 24 Hours After Birth)

Dopamine Norepinephrine Serotonin Females Males

C A C A C A C A

Mean Rate Constant/hr 0.354 0.380 0.261 0.497 0.853 0.852 0.573 0.740

Steady-State Concentration ii g/g Tissue 0.665 0.727 0.138 0.202 0.46 0 49 0.48 0.41

(mean+ S . E . M . ) 20.03S 20.041 20.009 3 . 0 0 7 +o.O2 +O.OZ z0.03 f p . 0 3

Synthesis Rate rnpmole/g/hr 1.54 1.80 0.305 0.593 2.32 2.36 1.55 1.72

(mean? s . E . M . ) 20.10 +o.z8 20,014 20 020 9.20 + a . l o 20.14 -0.13

Number of Animals Used 19 16 19 18 23 17 19 2 1


to the controls. JILEK et al. (1964) noted microscopic changes in the brains of neonatal rats within 24h after anoxic insult, but then found no evidence of lesions after maturation ; it was their suggestion that behavioral deficits might be due to a long-term alteration of metabolic systems that prompted our research. Because we were injecting the precursor amino acids into the general circulation via the tail- vein, we investigated the rate of transport of tyrosine and tryptophan into the brain (SIMON, 1974). We thought this might explain the increased activity of tyrosine and tryptophan in the brain 9Ck-120 min after injection; but there was no evidence of increased penetration of the precursor amino acids into the brain (SIMON, 1974).

We cannot relate the biochemical differences we have found to any neurobehavioral deficits, but we can conclude that an asphyxic insult in the neonatal period can affect metabolic systems of the brain without producing permanent, gross brain damage. Retar- dation of body growth and the greater growth lag in male animals suggest, in particular, a possible in- terference with development of neuroendocrine systems.

Sex dgerences in serotonin metabolism Sex differences in rat brain serotonin levels and

metabolism have been noted in several studies (KATO, 1960: SKILLEN et al., 1961; LADOSKY & GAZ~RI, 1970; GIULIAN et al., 1973; HARDIN, 1973). A higher serotonin synthesis rate in females had been suggested by SKILLEN et al. (1961) and HARDIN (1973) on the basis of in vitro studies of 5-hydroxytryptophan decarboxy- lase and monoamine oxidase; our finding of greater in viuo conversion of tryptophan to serotonin in female rat brain confirms this. Administration of testosterone, estrogens and castration have been found to affect the development of brain serotonin (LADOSKY & GAZIRI, 1970; GIULIAN et al., 1973). LADOSKY & GAZIRI ( I 9-70), studying the androgenizing effect of testosterone administration to female rats in the neonatal period, found a normal increase of forebrain and midbrain serotonin levels in females at 12 days of age; this increase was prevented by a single injection of testosterone propionate (0.1 mg.) on the first day of life. A comparable (though somewhat smaller) increase in brain serotonin levels at 12 days of age was found in castrated males.

Although biosynthesis and storage of monoamines is limited in the neonatal rat, probably the acute effect of anoxia or hypoxia is to deplete the monoamine stores of the brain (HURWITZ et al., 1971; CVMERMAN et al., 1972). Reserpine is known to deplete m o n e amine stores (BRODIE et al., 1959), and if reserpine is injected at the time of neonatal testosterone administration to female rats, the androgenizing effects on later development are prevented (LAWSKY & GAZIRI, 1970). A depletion of monoamines by asphyxic stress in the neonatal perod may selectively alter the development of serotonin metabolism only

in male animals by altering the normal interaction of testosterone with serotonin in the central nervous system. Growth retardation after neonatal asphyxia may result directly from monoamine depletion in the brain. Secretion of growth hormone from the pitui- tary appears to be initiated by serotonin or (more likely) a serotonin metabolite (SASSIN et al., 1969; COLLU et al., 1972); growth hormone release is also impaired after depletion of monoamine stores by reserpine administration (SACHAR et al., 1972). The apparently greater vulnerability of males to neonatal asphyxia suggests that the asphyxic insult had a greater effect on the testosterone mediated stages of postnatal development of the male brain and that the asphyxic insult had a lesser effect on estrogen mediated stages of brain development in female animals. Perhaps estrogens protect the female and pre- vent, or minimize the results of, asphyxic damage.

Maturation and stress The possibility exists that increased norepinephrine

synthesis after neonatal asphyxia, at least in some regions of the brain, may be due to maturational delay. PORCHER & HELLEX (1972) in an investigation of regional development of catecholamine synthesis in rat brain found that in this species, between 7 and 45 days of age, the tyrosine hydroxylase activity in several brainstem areas equalled or exceeded the adult rate. The activity of tyrosine hydroxylase in forebrain areas of immature animals is lower than in adult rats however and approaches the adult level of enzyme activity slowly, reaching only 75% of the normal adult rate by 45 days of age. Since only whole brain was assayed in the experiments after neonatal asphyxia presented here, it is not possible to say whether the increased synthesis rate found was due primarily to increased biosynthetic activity in subcortical or cortical regions.

HUTTUNEN (1971) observed an increase.in forebrain norepinephrine turnover in W 5 - d a y old rats after prenatal stress (maternal footshock). ADAMSONS et al. (1971) noted that maternal stress causes release of catecholamines, which interferes with uterine blood- flow and produces, in monkey offspring the same pattern of focal brainstem lesions found after a brief period of total asphyxia at birth. The stress produced by maternal footshock during pregnancy, then, may compromise the offspring in a way comparable to neonatal asphyxia. HUTTUNEN’S (1971) finding of increased norepinephrine turnover in the forebrain of prenatally stressed rats suggests that the effect is due less to immaturity than to premature induction of enzyme transcription and synthesis. This would corre- spond to the premature development of hepatic tryptophan oxygenase in rats reared in an hypoxic environment (FRANCESCONI & MAGER, 1969), or early induction of other hepatic enzymes found after prenatal or neonatal hormone injections (GREENGARD, 1969).


In conclusion, we cannot be sure that neonatal asphyxia in the rat provides a useful model for any human affliction. It is of possible significance that male animals were more vulnerable to neonatal asphyxia, as there is about a 4 : l greater preponder- ance of males over females in the human population who develop early schizophrenic illness, or infantile autism (WING, 1966). In one pilot experiment (SIMON, 1974) we found a decreased serotonin binding capacity of blood platelets from asphyxiated rats compared with control animals. A similar deficiency in scrotonin binding capacity has been reported for autistic children (BOUILLIN et al., 1970). The use of paired littermates in our experiments excludes the possibility of genetic causes underlying the differences we have found. This suggests that similar metabolic anomalies in human congenital disorders may also be due to factors other than genetic abnormalities.

Acknowledgements-This work was supported by a grant from the Benevolent Foundation of Scottish Rite Free- masonry, Northern Jurisdiction, U.S.A. (the Scottish Rite Schizophrenia Research Program). We are also grateful for support, for NICOLE SIMON: from the American Association of University Women, for the Connie M. Guion Fellow- ship for Medical Research in 1971-1972 and from the National Institute of Mental Health, for predoctoral fel- lowship number l F01 MH51868-01 for 1972-1974. The authors would like to thank Mr. B. HURTEX and Mr. R. WICKE (who was supported, in part, by U.R.O.P. Work- study Program of the Massachusetts Institute of Tech- nology) for their valuable technical assistance.

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Documents

NEONATAL ASPHYXIA IN THE RAT: GREATER VULNERABILITY OF MALES AND PERSISTENT EFFECTS ON BRAIN MONOAMINE SYNTHESIS