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Cerebral edema in developing brain: I. Normal water and cation content in developing rat brain and postmortem changes
EXPERIMENTAL
32, 431-438 (1971)
NEUROLOGY
Cerebral
Edema
Water Rat
Brain:
I. Normal
and
Cation
Content
in Developing
Brain
and
Postmortem
Changes
S. I$‘. Departrncrzt
in Developing
of Child
DE
Health,
SOUZA
AND
of
Ukcrsity Received
JOHN
May
DOBBING
Manchcstrv,
1 Marachester
13, England
21, 1971
In preparation for an experimental study of cerebral edema in immature brain, the normal development of rat brain has been studied with respect to brain weight, water, sodium, and potassium content from 16 days of gestation at frequent intervals until adult life. The time course of postmortem alteration of these values has been followed until 96 hr after death in the hope that brains from human autopsy may be assessed for the presence of edema. It has been noted that con-
siderable alterations
of composition
corresponding to the perinatal period of the differences between immature cerebral edema in babies is discussed.
occur at those stages of brain development in humans. and mature
The significance brain, for the
of these, assessment
and of
Introduction
Although many varieties of edema and swelling have been studied in adult brain (3, 6), there is comparatively little information about its nature and etiology in immature nervous tissue. Edema is often loosely referred to in human baby brain without any precise definition of criteria, while in the experimental animal it has been suggested that it is unlikely even to occur before a certain immature stage of brain growth (8, 9). The present paper is an account of normal rat brain growth in respect of its water, sodium, and potassium composition from the 16th day of gestation until maturity. Its purpose is to serve as a base line for studies of experimental edema of immature brain to be produced in a variety of different ways. A previous study (10) provided insufficient information for this purpose relating to the late fetal and early postnatal period. In addition postmortem
changes
in the level of these constituents
have been investigated,
1 This work was supported by a grant from the Medical Research Council. We are also grateful to the National Fund for Research into Crippling Diseases, to the Spastics Society for their help and to the Special Donations Fund of the Department of Child Health.
431
432
DE
SOUZA
AND
DOBBING
so that an attempt may be made to interpret findings in human material which is usually only available some hours after death. Materials
and
autopsy
Methods
Rats of a black hooded strain were used throughout. The day of mating was determined by examination of vaginal lavages for stage of estrus and presence of sperm. Under these conditions the length of gestation was 21-22 days. On the day of birth all litters were reduced to six male animals so that their subsequent rate of growth and development was standardized. Only litters containing at least six males were used, and if these did not all survive, the entire litter was rejected. All postnatal animals were analyzed individually. Animals were housed in well standardized conditions with regard to temperature, humidity, ventilation, and lighting. One hundred sixty-two fetuses and 10s postnatal animals were examined. The number at each fetal age is shown in Table 1. Six postnatal litter mates were killed at each age shown on the graphs. Fetal rats were obtained after killing the mother with an overdose of ether. Postnatal animals were killed by decapitation. In all cases the whole brain was immediately removed, precautions being taken to avoid evaporation or contamination with extraneous sodium or potassium. The surface of the brain was quickly washed free from blood and gently blotted before weighing. Homogenates were then prepared and aliquots taken into weighed bottles for determinations of water, sodium, and potassium content. Fetal brains were bulked with others from the same litter and homogenized together to provide enough material for analysis. Postnatal brains were treated individually. For the study of postmortem changes in brain water and electrolyte levels, 30 adult rats of the same strain weighing over 200 g were used. Six were killed and their brains removed within 3 min of death. The reTABLE NUMBERS
OF FETAL
RATS
I USED
AT EACH
AGE
a
Gestational age (days)
No. of animals
No. of bulked groups h
16 17 18 19 20 21
37 23 25 25 24 28
3 3 3 4 4 6
Q Six litter mates were analyzed at each postnatal age. b Each group came from the same litter.
CEREBRAL
EDEMA
IN
DEVELOPING
433
BRAIN
mainder were killed and their intact bodies kept first at room temperature (22-24 C) for 1 hr and then in a refrigerator (5 C) for various periods up to 96 hr after death before removing their brains for analysis. Three rats were examined at each interval of time after death. Water, sodium, and potassium content were estimated individually in each brain. Water was estimated by drying to constant weight at 100 C. Sodium and potassium were determined by flame photometry (7) after digesting the tissue with concentrated nitric acid. Results
and
Discussion
Brain Weight and Body Weight. The brain weights, body weights, and brain: body ratios of this series of normal rats from 16 days of gestation to 70 days of postnatal age are shown in Fig. 1. The unusually small variance at each age is an example of the degree of standardization which can
BIRTH
WEANING
I
0
1
4 AGE
6
0
IO
(weeks)
FIG. 1. Body weights (45), brain weights (47), and brain weight: body weight ratios (X) in developing rats. Standard deviations of less than 5 g body wt, 0.05 g brain wt, and 0.2% brain: body ratio are not shown. Numbers of animals are shown in Table 1.
434
DE
SOUZA
AND
DOBBING
be achieved in these parameters under the conditions of animal selection and environmental control described above. Figure 1 illustrates the precedence taken by the brain “growth spurt” over that of the body and the predominantly postnatal pattern of brain growth in this species. The decline in brain weight : body weight ratio which occurs prenatally and after weaning at 21 days is temporarily halted during the suckling period. In approximately the first 5 days of postnatal life the ratio shown in Fig. 1 may be somewhat lower than the true value, since at this age the stomach contains an amount of milk which is large in relation to the body weight and no attempt was made to correct for this. The period during which the brain weight: body weight ratio remains constant can be shortened or lengthened by varying the rate of bodily
0
4
2 AGE
6
8
d
IO
(weeks)
FIG. 2. Brain sodium, potassium, and sodium: potassium ratio in developing rats. Standard deviations of less than 1.25 mM/kg sodium and potassium, and less than 0.25 aodium: potassium ratio are not shown. Numbers of animals are shown in Table 1.
CEREBRAL
EDEMA
IN
DEVELOPING
435
BRAIN
growth during the suckling period. Under optimum conditions in fast growing litters of three the ratio begins to decline again after a very short postnatal pause lasting a few days (Dobbing and Sands, in preparation). Brain, Sodium, Potassium, apzd Water. Neither the brain sodium (mM/ kg fresh wt: Fig. 2) nor the brain water content (,% fresh wt; Fig. 3) undergo very much change prenatally nor in the first few postnatal days. The sodium then declines during the suckling period to reach an adult level soon after weaning. The water falls rather more slowly and is still above the adult level at weaning, declining slowly but steadily at least until 10 weeks of age. The fastest decline is from about 7 postnatal days to about 28 days. In all these respects the pattern of decline in brain water more closely follows the reciprocal of the increase in brain lipid concentration than the declining sodium content (Dobbing and Sands, in preparation). The lipid increase in turn follows the time course of myeIination. The brain potassium (mM/kg fresh wt; Fig. 2) follows a quite different pattern from either the water or the sodium. It falls steeply during the last week of fetal life, reaching its lowest level soon after birth. It then rises to a new peak during the fourth postnatal week and very slowly declines again. The influence of this on the brain sodium: potassium ratio is profound (Fig. 2). Rising to a peak soon after birth, the ratio falls to near-adult levels by the time of weaning and may even undergo a slow small rise subsequently.
d
I 2
I 4 AGE
FIG.
shown.
3. Brain Numbers
I 6
1 IO
9
(weeks)
water in developing rats. Standard of animals are shown in TabIe 1.
deviations
less than
0.3%
are not
436
DE
SOUZA
AND
DOBBING
Note that all the major changes in brain sodium, potassium, and water occur during the time of the brain growth spurt which happens to be postnatal in the rat. This is also the timing of the very active phase of oligodendroglial multiplication which precedes the synthesis of myelin lipids.
*OD
f
-0 (2’-
r=‘)
L13lV~
NIVYB
(SWW”‘)
WlMSVlOd
NIVYB
* .o
P
(a4/
WW) WIIIOOS NIV’YB
FIG. 4. Postmortem changes in brain water, sodium, and potassium in adult rats. Standard deviations of less than 0.25% water and 1.25 m&r/kg sodium and potassium are not shown. Each point is the mean of three animals. The range is shown where this was large.
CEREBRAL
EDEMA
IN
DEVELOPING
BRAIN
437
Both of these latter processes coincide with the period of growth of cell processes from neurons, most of which have already arrived at about the time of birth in this species (4) before the growth spurt begins. As might be expected, there are also dramatic developmental changes in the activity of several brain enzymes accompanying the structural changes of the growth spurt ( 1, 2). The comparable period in human brain development is much longer than in the rat, extending from about 30 weeks of gestation to at least 1s months of postnatal age (5). Changes After Death. Adult rats were used in this study so as to avoid variation between animals as much as possible. Figure 4 shows that brain sodium, potassium, and water change considerably in the first few hours after death under these conditions. Water is the first to reach a steady value, but it may be 48 hr before sodium and potassium do so. The blood circulation is arrested, and so the fall in potassium and rise in sodium probably represent a tendency to equilibrate with the very low potassium and high sodium of the cerebrospinal fluid compared with their levels in brain. Brain tissue fluid may have up to ten times the volume of the cerebrospinal fluid and this will affect the equilibrium position. Since sodium and potassium levels move in different directions postmortem, there are great alterations of the sodium: potassium ratio. Thus if any of these parameters are to be used for postmortem assessmentof cerebral edema in human brain it will be necessary to wait until equilibrium has been reached. Conclusions. The present results show that considerable individual variation can be eliminated from a seriesof developing rats by the rigorous control of environmental and other conditions of rearing employed here. Also, in the assessmentof experimental cerebral edema in the immature brain, great care will have to be taken to standardize the developmental age. This will especially be true for those parameters such as the ones reported here which are changing rapidly at the age studied. Pediatricians and pathologists should not use the expression “cerebral edema” loosely during early growth and development. It is going to be a very difficult diagnosis to substantiate in any human baby. References
1.
B. P. F., and J. DOBBIKG. 1971.Vulnerability of developingbrain: III. Developmentof four enzymesin the brainsof normaland undernourished rats.
ADLARD,
Brain Res. 28 : 97-107. B. P. F., and J. DOBBING. 1971.Phosphofructokinase and fumaratehydratasein developingrat brain. J. Nc~oclze~r~. 18: 1219-1303. BAKAY, L., and J. C. LEE. 1965.“CerebralEdema.”Thomas, Springfield, Ill. DAVISOX, A. N., and J. DOBBIKG. 1968.The developingbrain,pp. 253-286.In “Applied Neurochemistry.”A. N. Davison and J. Dobbing. [Eds.]. Blackwell,
2. ADLARD, 3. 4.
Oxford.
438
DE
SOUZA
AND
DOBBING
J. 1970. Undernutrition and the developing brain: the relevance of animodels to the human problem. Amer. J. Dis. Child. 120: 411-415. 6. KLATZO, I., and F. SEITELBERGER. 1967. “Brain Edema.” Springer-Verlag, New York. 7. MCILWAIN, H., and R. RODNIGHT. 1962. “Practical Neurochemistry.” Churchill, London. 8. SELLAR, M. J., and R. G. SPECTOR. 1963. Water induced cerebral overhydration in the maturing rat brain. Nature London 198 : 489-490. 9. SPECTOR, R. G. 1962. Water content of the immature rat brain following cerebral anoxia and ischaemia. Brit. J. Exp. Pathol. 43 : 472-479. 10. VERNADAKIS, A., and D. M. WOODBURY. 1962. Electrolyte and amino acid changes in the rat brain during maturation. Amer. J. Physiol. 203: 748-752. 5.
DOBBING,
mal
32, 431-438 (1971)
NEUROLOGY
Cerebral
Edema
Water Rat
Brain:
I. Normal
and
Cation
Content
in Developing
Brain
and
Postmortem
Changes
S. I$‘. Departrncrzt
in Developing
of Child
DE
Health,
SOUZA
AND
of
Ukcrsity Received
JOHN
May
DOBBING
Manchcstrv,
1 Marachester
13, England
21, 1971
In preparation for an experimental study of cerebral edema in immature brain, the normal development of rat brain has been studied with respect to brain weight, water, sodium, and potassium content from 16 days of gestation at frequent intervals until adult life. The time course of postmortem alteration of these values has been followed until 96 hr after death in the hope that brains from human autopsy may be assessed for the presence of edema. It has been noted that con-
siderable alterations
of composition
corresponding to the perinatal period of the differences between immature cerebral edema in babies is discussed.
occur at those stages of brain development in humans. and mature
The significance brain, for the
of these, assessment
and of
Introduction
Although many varieties of edema and swelling have been studied in adult brain (3, 6), there is comparatively little information about its nature and etiology in immature nervous tissue. Edema is often loosely referred to in human baby brain without any precise definition of criteria, while in the experimental animal it has been suggested that it is unlikely even to occur before a certain immature stage of brain growth (8, 9). The present paper is an account of normal rat brain growth in respect of its water, sodium, and potassium composition from the 16th day of gestation until maturity. Its purpose is to serve as a base line for studies of experimental edema of immature brain to be produced in a variety of different ways. A previous study (10) provided insufficient information for this purpose relating to the late fetal and early postnatal period. In addition postmortem
changes
in the level of these constituents
have been investigated,
1 This work was supported by a grant from the Medical Research Council. We are also grateful to the National Fund for Research into Crippling Diseases, to the Spastics Society for their help and to the Special Donations Fund of the Department of Child Health.
431
432
DE
SOUZA
AND
DOBBING
so that an attempt may be made to interpret findings in human material which is usually only available some hours after death. Materials
and
autopsy
Methods
Rats of a black hooded strain were used throughout. The day of mating was determined by examination of vaginal lavages for stage of estrus and presence of sperm. Under these conditions the length of gestation was 21-22 days. On the day of birth all litters were reduced to six male animals so that their subsequent rate of growth and development was standardized. Only litters containing at least six males were used, and if these did not all survive, the entire litter was rejected. All postnatal animals were analyzed individually. Animals were housed in well standardized conditions with regard to temperature, humidity, ventilation, and lighting. One hundred sixty-two fetuses and 10s postnatal animals were examined. The number at each fetal age is shown in Table 1. Six postnatal litter mates were killed at each age shown on the graphs. Fetal rats were obtained after killing the mother with an overdose of ether. Postnatal animals were killed by decapitation. In all cases the whole brain was immediately removed, precautions being taken to avoid evaporation or contamination with extraneous sodium or potassium. The surface of the brain was quickly washed free from blood and gently blotted before weighing. Homogenates were then prepared and aliquots taken into weighed bottles for determinations of water, sodium, and potassium content. Fetal brains were bulked with others from the same litter and homogenized together to provide enough material for analysis. Postnatal brains were treated individually. For the study of postmortem changes in brain water and electrolyte levels, 30 adult rats of the same strain weighing over 200 g were used. Six were killed and their brains removed within 3 min of death. The reTABLE NUMBERS
OF FETAL
RATS
I USED
AT EACH
AGE
a
Gestational age (days)
No. of animals
No. of bulked groups h
16 17 18 19 20 21
37 23 25 25 24 28
3 3 3 4 4 6
Q Six litter mates were analyzed at each postnatal age. b Each group came from the same litter.
CEREBRAL
EDEMA
IN
DEVELOPING
433
BRAIN
mainder were killed and their intact bodies kept first at room temperature (22-24 C) for 1 hr and then in a refrigerator (5 C) for various periods up to 96 hr after death before removing their brains for analysis. Three rats were examined at each interval of time after death. Water, sodium, and potassium content were estimated individually in each brain. Water was estimated by drying to constant weight at 100 C. Sodium and potassium were determined by flame photometry (7) after digesting the tissue with concentrated nitric acid. Results
and
Discussion
Brain Weight and Body Weight. The brain weights, body weights, and brain: body ratios of this series of normal rats from 16 days of gestation to 70 days of postnatal age are shown in Fig. 1. The unusually small variance at each age is an example of the degree of standardization which can
BIRTH
WEANING
I
0
1
4 AGE
6
0
IO
(weeks)
FIG. 1. Body weights (45), brain weights (47), and brain weight: body weight ratios (X) in developing rats. Standard deviations of less than 5 g body wt, 0.05 g brain wt, and 0.2% brain: body ratio are not shown. Numbers of animals are shown in Table 1.
434
DE
SOUZA
AND
DOBBING
be achieved in these parameters under the conditions of animal selection and environmental control described above. Figure 1 illustrates the precedence taken by the brain “growth spurt” over that of the body and the predominantly postnatal pattern of brain growth in this species. The decline in brain weight : body weight ratio which occurs prenatally and after weaning at 21 days is temporarily halted during the suckling period. In approximately the first 5 days of postnatal life the ratio shown in Fig. 1 may be somewhat lower than the true value, since at this age the stomach contains an amount of milk which is large in relation to the body weight and no attempt was made to correct for this. The period during which the brain weight: body weight ratio remains constant can be shortened or lengthened by varying the rate of bodily
0
4
2 AGE
6
8
d
IO
(weeks)
FIG. 2. Brain sodium, potassium, and sodium: potassium ratio in developing rats. Standard deviations of less than 1.25 mM/kg sodium and potassium, and less than 0.25 aodium: potassium ratio are not shown. Numbers of animals are shown in Table 1.
CEREBRAL
EDEMA
IN
DEVELOPING
435
BRAIN
growth during the suckling period. Under optimum conditions in fast growing litters of three the ratio begins to decline again after a very short postnatal pause lasting a few days (Dobbing and Sands, in preparation). Brain, Sodium, Potassium, apzd Water. Neither the brain sodium (mM/ kg fresh wt: Fig. 2) nor the brain water content (,% fresh wt; Fig. 3) undergo very much change prenatally nor in the first few postnatal days. The sodium then declines during the suckling period to reach an adult level soon after weaning. The water falls rather more slowly and is still above the adult level at weaning, declining slowly but steadily at least until 10 weeks of age. The fastest decline is from about 7 postnatal days to about 28 days. In all these respects the pattern of decline in brain water more closely follows the reciprocal of the increase in brain lipid concentration than the declining sodium content (Dobbing and Sands, in preparation). The lipid increase in turn follows the time course of myeIination. The brain potassium (mM/kg fresh wt; Fig. 2) follows a quite different pattern from either the water or the sodium. It falls steeply during the last week of fetal life, reaching its lowest level soon after birth. It then rises to a new peak during the fourth postnatal week and very slowly declines again. The influence of this on the brain sodium: potassium ratio is profound (Fig. 2). Rising to a peak soon after birth, the ratio falls to near-adult levels by the time of weaning and may even undergo a slow small rise subsequently.
d
I 2
I 4 AGE
FIG.
shown.
3. Brain Numbers
I 6
1 IO
9
(weeks)
water in developing rats. Standard of animals are shown in TabIe 1.
deviations
less than
0.3%
are not
436
DE
SOUZA
AND
DOBBING
Note that all the major changes in brain sodium, potassium, and water occur during the time of the brain growth spurt which happens to be postnatal in the rat. This is also the timing of the very active phase of oligodendroglial multiplication which precedes the synthesis of myelin lipids.
*OD
f
-0 (2’-
r=‘)
L13lV~
NIVYB
(SWW”‘)
WlMSVlOd
NIVYB
* .o
P
(a4/
WW) WIIIOOS NIV’YB
FIG. 4. Postmortem changes in brain water, sodium, and potassium in adult rats. Standard deviations of less than 0.25% water and 1.25 m&r/kg sodium and potassium are not shown. Each point is the mean of three animals. The range is shown where this was large.
CEREBRAL
EDEMA
IN
DEVELOPING
BRAIN
437
Both of these latter processes coincide with the period of growth of cell processes from neurons, most of which have already arrived at about the time of birth in this species (4) before the growth spurt begins. As might be expected, there are also dramatic developmental changes in the activity of several brain enzymes accompanying the structural changes of the growth spurt ( 1, 2). The comparable period in human brain development is much longer than in the rat, extending from about 30 weeks of gestation to at least 1s months of postnatal age (5). Changes After Death. Adult rats were used in this study so as to avoid variation between animals as much as possible. Figure 4 shows that brain sodium, potassium, and water change considerably in the first few hours after death under these conditions. Water is the first to reach a steady value, but it may be 48 hr before sodium and potassium do so. The blood circulation is arrested, and so the fall in potassium and rise in sodium probably represent a tendency to equilibrate with the very low potassium and high sodium of the cerebrospinal fluid compared with their levels in brain. Brain tissue fluid may have up to ten times the volume of the cerebrospinal fluid and this will affect the equilibrium position. Since sodium and potassium levels move in different directions postmortem, there are great alterations of the sodium: potassium ratio. Thus if any of these parameters are to be used for postmortem assessmentof cerebral edema in human brain it will be necessary to wait until equilibrium has been reached. Conclusions. The present results show that considerable individual variation can be eliminated from a seriesof developing rats by the rigorous control of environmental and other conditions of rearing employed here. Also, in the assessmentof experimental cerebral edema in the immature brain, great care will have to be taken to standardize the developmental age. This will especially be true for those parameters such as the ones reported here which are changing rapidly at the age studied. Pediatricians and pathologists should not use the expression “cerebral edema” loosely during early growth and development. It is going to be a very difficult diagnosis to substantiate in any human baby. References
1.
B. P. F., and J. DOBBIKG. 1971.Vulnerability of developingbrain: III. Developmentof four enzymesin the brainsof normaland undernourished rats.
ADLARD,
Brain Res. 28 : 97-107. B. P. F., and J. DOBBING. 1971.Phosphofructokinase and fumaratehydratasein developingrat brain. J. Nc~oclze~r~. 18: 1219-1303. BAKAY, L., and J. C. LEE. 1965.“CerebralEdema.”Thomas, Springfield, Ill. DAVISOX, A. N., and J. DOBBIKG. 1968.The developingbrain,pp. 253-286.In “Applied Neurochemistry.”A. N. Davison and J. Dobbing. [Eds.]. Blackwell,
2. ADLARD, 3. 4.
Oxford.
438
DE
SOUZA
AND
DOBBING
J. 1970. Undernutrition and the developing brain: the relevance of animodels to the human problem. Amer. J. Dis. Child. 120: 411-415. 6. KLATZO, I., and F. SEITELBERGER. 1967. “Brain Edema.” Springer-Verlag, New York. 7. MCILWAIN, H., and R. RODNIGHT. 1962. “Practical Neurochemistry.” Churchill, London. 8. SELLAR, M. J., and R. G. SPECTOR. 1963. Water induced cerebral overhydration in the maturing rat brain. Nature London 198 : 489-490. 9. SPECTOR, R. G. 1962. Water content of the immature rat brain following cerebral anoxia and ischaemia. Brit. J. Exp. Pathol. 43 : 472-479. 10. VERNADAKIS, A., and D. M. WOODBURY. 1962. Electrolyte and amino acid changes in the rat brain during maturation. Amer. J. Physiol. 203: 748-752. 5.
DOBBING,
mal