Effects of hypothyroidism on the differentiation of neurons and glia in developing rat cerebrum

Journal o! the neurological Sciences, 18 (1973) 155-173 .{ Elsevier Scientific Publishing Company, Amsterdam

155 Printed in The Netherlands

Effects of Hypothyroidism on the Differentiation of Neurons and Glia in Developing Rat Cerebrum* NORMAN H. BASS** AND ELIZABETH YOUNG Departments ~] Neurology and Pharmacology, University of' Virginia School ~]" Medicine, Charlottesville, Virginia (U.S.A.) (Received 31 July, 1972)

INTRODUCTION

Hypothyroidism during the perinatal period results in permanent alterations of brain and body growth in man (Bacon, Lowrey and Carr 1967; Smith, Blizzard and Wilkins 1957; Topper 1951) and experimental animals (Eayrs 1964, 1966; Myant 1965). In newborn rats, surgical removal of the thyroid gland requires great technical skill and is accompanied by a relatively high operative mortality (Scow and Simpson 1945). Alternatively, complete atrophy of the thyroid gland in the newborn rat can be produced, with negligible mortality, by a single intraperitoneal injection of radioactive iodine (Goldberg and Chaikoff 1949 ; Hamburgh 1968). This latter technique selectively destroys functioning thyroid tissue in 12-24 hr, and decreases serum levels of thyroid hormone by 87 ~ within 3 days (Geloso, Hereon, Legrand and Jost 1968; Samel 1968). Rats subjected to thryoidectomy at birth show a striking impairment in their rate of body growth, resulting in a dwarfed adult with retarded behavioral development (Eayrs and Lishman 1953), and impaired learning ability (Eayrs and Levine 1963). The cerebral cortex of such animals exhibits histologic changes which include abnormal organization of the capillary bed (Eayrs 1954), decreased size of neuronal perikarya (Eayrs and Taylor 1951), diminished growth of the neuropil (Eayrs 1955; Eayrs and Horn 1955), and reduced deposition of myelin (Barrnett 1950). In addition, neurochemical studies have delineated abnormalities of protein metabolism (Cocks, Balazs, Johnson and Eayrs 1970; Geel, Valcana and Timiras 1967; Ramirez and Gomez 1966), enzyme activity (Geel and Timiras 1967 ; Hamburgh and Flexner 1957; Valcana and Timiras 1969), lipid composition (Balazs, Brooksbank, Davison, Eayrs and Wilson 1969; Myant 1965; Walravens and Chase 1969), and nucleotide content

*This investigation was supported by Public Health Service Research Grants No. NS-07563. Institution Subgrant No. 2707-0204 and National Science Foundations Grants No. GB 98 201 and Institutional Subgrant No. 71-268. ** John and Mary R. Markle Scholar in Academic Medicine,

156

N . H . BASS, E. YOUNG

(Balazs, Kovacs, Teichgraber, Cocks and Eayrs 1968; Geel et al. 1967: Pasquini~ Kaplun, Carcia and Gomez 1967). Although these prior biochemical studies of whole brain or entire cerebral cortex are of interest, their value in describing the neurochemical pathology of cerebral cortex resulting from decreased thyroid hormone is limited because the maturation of the various regions of brain is asynchronous. Therefore, we have confined our observations to the somatosensory area of the cerebrum in rats made hypothryoid at birth, combining microchemical techniques with histologic examination to study abnormalities in cellular differentiation.

MATERIALS AND METHODS

Production of neonatal hypothyroidism Ten pregnant rats of the Charles River CD ® strain, at 14 to 16 days of gestation, were fed ad libitum on a standard laboratory rodent diet. Parturition occurred on days 21 to 23. Sixty ariimals from these litters were used, and each litter was reduced to 8 pups at birth. Five litters formed the control group, and the other 5 litters were made hypothyroid within 8 hr of birth by giving each pup a single intraperitoneal injection of 150 ~Ci of carrier-free iodine-131 dissolved in normal saline. The volume of solution injected into each newborn animal was calculated from the decay curve of the isotope and equaled 50/~l. Separate experimental and control litters were established since it was discovered that hypothyroid animals were unable to compete with control littermates for their mother's milk. Moreover, mothers were allowed to nurse their offspring, continuously until 35 days of age in addition to providing ad libitum intake of standard diet to insure maximal nutrition. After weaning, the animals were segregated by sex, placed in separate cages and fed on a standard rodent diet. Offspring were weighed at birth and daily thereafter. Both the normal and hypothyroid animals were divided into 5 groups, then killed by decapitation when 10, 20, 30, 40, and 50 days old. An additional group was examined immediately after birth, prior to thyroidectomy, in order to obtain further details of accumulation of DNA, RNA, total proteins and ganglioside sialic acid. Each group contained 5 rats except the 50-day-old control group, which had 10 rats. Microchemical samplin9 and analysis After sacrifice, the brains were removed immediately, and the left cerebral hemisphere was placed on a glass slide and stored in a plastic specimen tube at - 7 0 ° C. The procedures for sampling a frozen block for quantitative microchemical assay of cerebral cortex and white matter and for relating the results to the microscopic appearance have been described in detail (Bass, Netsky and Young 1969)i Briefly, the frozen block of left cerebrum was sampled in a Harris cryostat at - 12° C ; 2 cylinders, each 2 mm in diameter, were punched perpendicular to the cortical surface of the block. The first frozen cylinder was measured with a micrometer caliper to determine the cortical depth, then placed in a stoppered weighing bottle and brought to room temperature in 10 min. The wet weight was obtained on a Mettler M-5 microbalance. The mean standard errors (+ 9 ~) for wet weight were large, reflecting variability produced by inconstant equilibration between tissue water and the atmosphere. The

EFFECTS OF HYPOTHYROIDISM ON DIFFERENTIATION OF NEURONS AND GLIA

157

TABLE 1 TO IAL SOLIDS, W A T E R C O N T E N T A N D C O R T I C A L T H I C K N E S S IN C E R E B R U M OF D E V E L O P I N G H Y P O T H Y R O I D RATS

Age (days)

Specimen

Fresh weight (#g/mm3) "

0 10

Normal Hypothyroid Normal Hypothyroid Normal Hypothyroid Normal Hypothyroid Normal Hypothyroid Normal

896 ± 59 770 ± 80 980 ± 85 761 ± 54 962 ± 83 916 ± 48 982 ± 94 770 + 62 1133 ± 93 952±65 1000±94

20 30 40 50

Dry weit4ht (#~4/mm~) "

116 ± 3.3 90 + 8.5 122 _+3.7 145 ± 8.6 178 ± 5.2 189 + 8.6 206 ± 6.5 172 + 15, 260 +_7.8 200± 12 277±8.3

Water content ( % oJjresh ~eiyht) °

87 88 86 81 81 79 79 78 78 79 72

Cortical thickness (ram)" 0.87 1,62 1.65 1.62 1.72 1.62 1.75 1.62 1.75 1.62 i.78

" Values are the means of 5 determinations with standard errors of the means in 5 animals.

specimen was then placed in an oven at 105 ° C for 2 hr, further desiccated with a vacuum pump for 2 hr, and the dry weight determined. Values were calculated as dry and wet weight per mm 3 of fresh frozen tissue (Table 1). The second frozen cylinder was cut serially with a rotary microtome into horizontal sections 40 It in thickness. These slices were desiccated over calcium chloride and used in consecutive groups (usually 9) of 6 sections each. Each set of slices was weighed on a Mettler UM-7 ultramicrobalance and placed in tubes grouped into two anatomic categories based on the depth of the sample : cortex and subcortical white matter. The analytic scheme of Hess was used for multiple biochemical analyses on single microsamples (Hess and Lewin 1965; Hess and Thalheimer 1965). Each group of slices was extracted with chloroform-methanol-water (16:8:1, v/v/v) and partitioned with 0.015 M aqueous KC1. Ganglioside sialic acid was determined on the upper aqueous phase by the fluorometric method of Hess and Rolde (1964). Cerebrosides (including the sulfate esters) were determined on the washed lower phase by the orcinol-sulfuric acid reaction adapted to microscale by Hess and Lewin (1965); cholesterol, by the spectrophotometric method of Glick, Fell, and Sjolin (1964) ; and proteolipid proteins by the spectrophotometric method of Lowry, Rosebrough, Farr and Randall (1951). On the residue insoluble in chloroform-methanol, RNA was assayed by the ultraviolet absorption method of Ogur and Rosen (1950), DNA by the fluorometric method of Kissane and Robins (1958), and residue protein by the method of Lowry et al. (1951). Total proteins were calculated as the sum of residue and proteolipid proteins: total lipids were calculated as the difference between dry weight and total proteins. This latter calculation tends to overestimate the true value of total lipids as it includes small amounts of carbohydrate, nucleic acid and low molecular weight extractives. Mean values for somatosensory gray and white matter were calculated from each series of determinations, and expressed in terms of dry weight. These data were then multiplied by values for total solids in cortical cylinders at corresponding developmental ages and thus expressed as/~g per mm 3 of fresh frozen

White matter

Cortex

White matter

Cortex

White matter

Cortex

White matter

Cortex 51.8 (56.2) 45.5 (57.7) 38.6 (53.8) 68.9 (40.3) 66.7 (36.7) 78.8 (41.2_+1.7) 82.3 (32.7_+1,l)

(58.4)

(104)

74.2 (100) 85.9 (130) 72.9 (11t) 119 (105) 115 (95.5) 158 (114_+4.8) 121 (90.5 ~ 3,2)

(39.3) 53.2 (54.0) 45.3 (55.6) 58.6

°/o dr)' weight

(44.9) 47.9 (65.8) 45.0 (68.4) 85:0

flg/'mm 3

Total proteins

DNA, R N A

TABLE 2

0.35 (0.95) 0.43 (0.66) 0.27 (0.78} 0.25 (1.24) 0.35 (1.34) 0.80 (I.56+0.04) 0,76 (1.35_+0.0t)

(0.85)

(1.87) 0.28 0.43) 0.26 (0.49) 0.38

12g/mm3

(293) 43.4 (67.2) 40.9 (76.6) 59.4 (133) 54,7 (148) 67.5 (104) 42.3 (122) 39.1 (194) 54.4 (209) 12.6 (245_+7.2) 118 (211_+2.3)

"cells x 10 ~ mm 3

DNA

(4.86) 2.02 (5.14) 4.02 5.65_+0:16) 3.79 (4,87 _+0;05t

1.46

(3.81)

1.43

(5.34) 2.29 {3.24)

(4.01) 2.62 (4.76) 2.42

(3.52) 2.91

(15.7) 3.08

#g/mg dr), weight

0.87 (0.72) 0.81 (0.98 _+0.01) 0.85 (0.'71 -(~0.09)

(0.98)

(1.61) 0.70 (1.02) 0.90 (0.87) 0.94 (1.06) 1.04 (0,92) 0.85 (1.02) 0.99 (0.87) 0.70

l~g/mm 3

RNA

p#g/mm 3

(5.49) (0.74) 16.2 0.67 (15.2)" (0.64) 21.9 0.66 (11.3) (0.75) 15.9 1.84 (8.03) (2.62) 19.0 1.65 (6.2l) (2.36) 12.5 0.86 {9.76 (1.36) 23.5 0.88 (7.10 (1.32) 18.0 0.91 (5.09 (1.47) 16.0 0.96 (3.46 (1.04) 6.47 1.26 (4.06 (1.86_+0.08) 7.15 1.28 (3.39 (1.09+_0.06)

ttg,cell

(6.18) 7.47 (5.28) 7.37 (6.14) 12.7 (14.7) 11.4 (13.3) 4.58 (6.60) 4.64 (6.41) 5.32 (5.64) 5.56 (3.99) 6.28 (6.73_+0.31) 6.43 (393+0.22t

/tg:mg dry weiqht

Ganglioside sialic acid

AND GANGLIOSIDES IN DEVELOPING RAT CEREBRUM FOLLOWING NEONATAL HYPOTHYROIDISM

(8.17) 50.1 (36.7) 52.4 (31.7) 100 (64.3) 97.8 (51.4) 41.4 (42.4) 67.0 (35.0) 75.7 (24.6) 56.9 (16.1) 32.3 (24~6) 35,0 (t6.7)

p mole,cell

The data reported are mean values for hypothyroid and normal groups, of 5 animals each Values for age-matched m)ImaI animals are given m parentheses : slandard errols of the means are calculated only for the 50-day-old normal group, comprising 10 rats. "A diploid cell nucleus was assumed to contain 6.4 × 10 L2 g DNA,

50

40

30

20

Cortex Cortex

0 10

White matter

Specimen

Age (days)

TOTAL PROTEINS,

~< ©

>

z Z

White matter

Cortex

White matter

Cortex

White matter

Cortex

White matter

Cortex

White matter

Cortex

Specimen o~ dry weight

46.8 (46.0) 50.0 (44.4) 41.4 (41.6) 48.8 (43.8) 54.5 (36.6) 61.4 (46.2) 31.0 (59.7) 33.3 (63.3) 21.2 (58.8_+2.6) 39.6 (67.3_+2.4)

tly/mm 3

42.1 (56.4) 45.0 (54.3) 60.0 (74.1) 70.8 (78.0) 103 (75.2) 116 (95.1) 53.4 (155) 57.3 (164) 42.3 (162 +7.0) 79.2 (186_+6.5)

Total lipids

2.51 (4.38) 2.71 (5.02) 3.61 (8.06) 3.15 (11.9) 6.01 (12.0) 5.29 (17.1) 8.02 (28.0) 8.16 (39.8) " 6.18 (26.8+1.6) 6.50 (41.0_+1.8)

;l@/'mm3

2.78 (3.58) 3.01 (4.10) 2.49 (5.03) 2.17 (6.67) 3.18 (5.87) 2.80 (8.31) 4.66 (10.9) 4.74 (15.3) 3.09 (9.66_+0.60) 3.25 (14.8_+0.95)

i.~, dry weight

Cerebrosides

4.26 (4.89) 4.44 (7.87) 2.88 (6.27) 2.62 (4.31) 6.10 (8.21) 6.65 (10.6) 10.4 (17.3) 10.8 (23.6) 3.26 (17.3_+0.76) 4.12 (33.0_+1.2)

#.q/mm 3

_

oj /o dry wei#ht

4.73 (3.98) 4.93 (6.40) 1.99 (3.52) 1.80 (2.42) 3.23 (3.98) 3.52 (5.16) 6.04 (6.62) 6.30 (9.08) 1.63 (6.25+0.28) 2.06 (11.9_+0.42)

Cholesterol .

_

3.09 (1.82) 2.35 (2.41) 3.91 (2.93) 3.32 (3.68) 3.23 (3.28) 3.74 (5.57) 5.56 (5.86) 5.25 (8.90) 6.57 (9.82+0.90) 6.32 (14.1_+2.0)

kt.q/nlm3

m

_

3.43 (1.50) 2.61 (1.97) 2.70 (1.68) 2.29 (2.07) 1.71 (1.60) 1.98 (2.70) 3.23 (2.26) 3.05 (3.42) 3.28 (3.50+0.32) 3.16 (5.10+0.73)

J°~odry weight

Proteolipid proteins

The data reported are mean values for hypothyroid and normal groups, of 5 animals each. Values for age-matched normal animals are given in parentheses ; standard errors of the means are calculated only for the 50-day-old normal group, comprising 10 rats.

50

40

30

20

10

(days)

Age

TABLE 3

T O T A L LIP1DS, ('EREBROSII)ES, C H O L E S T E R O L A N D P R O T E O L O P 1 D P R O T E I N S IN D E V E L O P I N G R A F C E R E B R U M F O L L O W I N G N E O N A T A L t l Y P O T H Y R O I D I S M

0

~Z © ~, Zm ~ © o~ Z

~m :Z

© Z ~_

0 ~

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160

N . H . BASS. E. YOUNG

tissue. The rationale for this calculation in analyzing biochemical data from developing cerebrum has been discussed (Bass et al. 1969). Mean biochemical values in both hypothyroid and control animals, expressed as ~g/mm 3 of fresh frozen tissue, from birth to 50 days of age are shown in Tables 2 and 3 and represented graphically as percentages of normal 50-day-old values to compare the rates of accumulation of biochemical components in cortex and subcortical white matter. Microscopic studies

The right cerebral hemisphere and the neck region were dissected in each animal and tissues were placed in 10 ~ formalin. Coronal sections of the right somatosensory area of cerebrum were stained with luxol fast blue cresyl violet, and by the methods of Nissl and Bodian. These methods allowed histologic study of myelin, neurons, glia and processes of nerve cells. In addition, serial sections were cut through the laryngeal tissues, and stained with hematoxylin and eosin for examination of the thyroid gland. RESULTS

The most rapid rate of body growth of the albino rat normally occurs between 10 and 40 days of age. Rats made hypothyroid at birth and allowed unrestricted intake of food failed to show the normal growth spurt, achieving only 40 °~;i of the body weight of age-matched control animals between 10 and 50 postnatal days (Fig. 1). Although no mortality was noted during early postnatal life, between 20 and 30 days,

270- D ~

RELATIONSHIP OF AGE TO BODY WEIGHT

o~e NORMAL o---,o HYPOTHYROI

240210180--

150-120--

~

~a j~ ~p tq. ~o,,.,.,~..,°

90-60-

0

10

20

30 40 50 POSTNATALDAY

60

75

Fig. 1. Growth curve of normal male albino rats compared to that of rats tnade hypothyroid at birth. Each point represents the mean body weight of 30 animals, expressed in grams. Bracketed vertical lines on the normal curve represent the standard error of the means.

EFFECTS OF HYPOTHYROIDISM ON DIFFERENTIATION OF NEURONS AND GLIA

161

just preceding the normal period of maximum body growth, sudden death occurred in 20 ~o of the experimental animals. The cause of death was not evident on postmortem examination and was presumed to be metabolic.

H istolo.qic study Serial sections cut through the laryngeal tissues of animals treated with r~adioactive iodine showed either no trace of thyroid tissue or a gland that had been replaced by connective tissue in which no colloid was seen. The parathyroid glands were normal. At birth, the somatosensory cortex of both normal and hypothyroid rats is 900 ~ thick and contains densely-packed, undifferentiated neurons; silver-impregnated fibers and stainable myelin are absent. At 10 days of age, the cortices of both normal and experimental animals had mean widths of 1600/~. The 6 layers were well defined, and cellular packing density decreased compared to the cortex immediately after birth. Between 10 and 40 days of age, cortical width in control specimens increases by 100 ~t associated with increasing cell density. In hypothyroid rats, however, cortical measurements remained constant during this period, resulting in values which were 10 °/.. lower in experimental animals at 40 and 50 days of age (Table 1).Moreover, in

~umBiw

Normal

J

Hypothyroid

Fig. 2. Subcortical white matter in a 50-day-old normal rat and in an animal made hypothyroid at birth. In the hypothyroid rat, the loss of myelin is striking, the glial cells are irregular in shape and immature, and many cells with hyperchromatic cytoplasm lie deep in white matter near the ependymal surface of the lateral ventricle (× 1010, Luxol fast blue cresyl violet).

162

N . H . BASS, E. YOUNG

the cortex of hypothyroid animals, the cellular density did not increase and the cytoarchitecture became progressively disorganized. The cytoplasm of many neurons was hyperchromatic; axodendritic fibers in cortex were poorly impregnated with silver; and the number of glial cells was greatly reduced. The ultimate effecT.sof neonatal hypothyroidism on myelination were best seen in subcortical white matter: (1) myelinated fibers stained poorly with luxol fast blue (2) the normal pattern of cellular alignment was disturbed, and there was a marked decrease in the number of glial cells. In the subependymal germinal zone near the lateral ventricles, undifferentiated glial cells, presumably destined to migrate through white matter into cerebral cortex normally disappear by 20 postnatal days, but were still visible in specimens from 50day-old hypothyroid rats (Fig. 2). Wet weiyht, total solids, and water content (Table l ) In the cerebrum of normal rats, total solids increase by 52 ~o between 10 and 30 days, and by 34 ~ from 30 to 50 days of age. These increases are accompanied by 7'?,~, decrements in water content. The initial increment of total solids in hypothyroid animals was parallel to controls except for a 22 ~, decrease between birth and 10 days, Between 30 and 50 days, hypothyroid rats showed only slight increases ha total solids, resulting in values that were 28 o/o lower than 50-day-old controls. No abnormality of water content was found. DNA (Table 2, Fiyure 3) Between birth and 10 days of age, DNA in the cerebrum of both normal and hypo-

DNA CORTEX

WHITE MATTER

:

0 m

< ~E 0 Z 0

~ NORMAL

0----0

<

HYPOTHYROID

160-

140-

I t

120I00-

E

80-

•

6o-

0

10

20

30

40

50

10

20

30

40

50

POSTNATAl. DAY

Fig, 3. D N A in developing cortex and white matter of normal and hypothyroid rats. All values are expressed as percentage of D N A in normal specimens at 50 post-natal days.

EFFECTS OF HYPOTHYROIDISM ON DIFFERENTIATION OF NEURONS AND GLIA

163

thyroid animals decreased by 77 ~ and 85 ~o, respectively. However, the subsequent increases to adult values, normally found between 10 and 50 days in cortex and between 10 and 40 days in white matter, did not occur in hypothyroid rats. As a result, at 50 days DNA was 49 O/~olower in cortex and 44 °/o in white matter than in age-matched controls.

Ganqlioside sialic acid (Table 2, Fiyure 4) In the normal animals, total ganglioside sialic acid concentrations peak significantly in both gray and white matter at 20 days of age. Although hypothyroid rats showed a similar accumulation profile, peak values at 20 days were decreased by 30~, in both cortex and white matter. This peak at 20 days in the cortex of hypothyroid rats was followed by a progressive decline so that values at 50 days were 32'~'~, lower than in controls. White matter of hypothyroid rats at 20 days also showed a decrease in peak values for ganglioside sialic acid ; but between 30 and 50 days, ganglioside continued to accumulate so that adult values were 15 ~ greater than in controls.

GANGLIOSIDE 220-

CORTEX

WHITE MATTER

'2;;,0,

200I a

SIALIC ACID

180~

o

160-

..J

140-

~E oe 0 Z

120-

I0080-

d

60-

~--a"

40200

10

2'0

3'0

4'0

5'0

10

20

30

40

50

POSTNATAL DAY

Fig. 4. Ganglioside sialic acid in developing cortex and white matter of normal rats and of those made hypothyroid at birth. All values are expressed as percentage of ganglioside sialic acid in normal specimens at 50 post-natal days.

RNA (Table 2, Figure 5) During normal development, RNA decreases 37 ~o in cerebral cortex between birth and 10 days of age, and is then relatively constant up to 50 days. In white matter at 10 postnatal days, normal values are 25 ~ greater than in adults, but then fall after 30 days. Total RNA in hypothyroid animals decreased by 57 ~ in the cerebral cortex

164

N . H . B A S S , E. Y O U N G

RNA

2°°r

¢ ; NORMAL 0-----,0 HYPOTHYROID

CORTEX

WHITEMATTER

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i

120Z

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60-

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2~

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~0

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POSINATAL DAY

Fig. 5. RNA in developingcortexand whitematterof normalandhypothyroidrats.All values are expressed as percentageof RNA in normalspecimensat 50 post-natal days. between birth and 10 days and then remained at levels lower than control values during the later stages of development. In the white matter of hypothyroid animals, RNA was essentially normal at 10 days of age, then slightly declined but remained consistently higher than age-matched controls between 20 and 50 days.

Total proteins (Table 2) During normal postnatal development, total proteins progressively increase in cortex and white matter up to 30 days and then decline. During this period, hypothyroid rats showed a decreased rate of protein accumulation; but between 30 and 50 days, protein concentrations rose significantly, reaching values 40 °)/o greater in cortex than in controls at 50 days. A similar biochemical profile for the accumulation of total protein was found in white matter. Total lipids, cerebrosides, cholesterol and proteolipid proteins ( Table 3, Fig. (~; At 10 and 20 days of age, the concentrations of total lipids in gray and white matter were similar in both control and hypothyroid animals. By 30 days, values in animals made hypothyroid at birth were increased 27 ~ in cortex and 18 ~o in white matter compared to age-matched controls. Between 30 and 40 days, when total lipids double in cortex and white matter of normal animals, the concentrations in the cerebrum of hypothyroid rats were reduced by one-half. Between 40 and 50 days of age, total lipids further decreased in cortex and showed only a slight rise in white matter of hypothyroid rats, resulting in concentrations 72 ~ and 57 ~o respectively, below normal 50day-old values.

EFFECTS OF HYPOTHYROIDISM ON DIFFERENTIATION OF NEURONS AND GLIA :

NORMAL

o.-~.-o

165

HYPOTHYROID

CEREBROSIDES

TOTAL LIPID 10o-

o

90 gO-

~c 70 60,~

50-

:E

~/j

40-

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CHOLESTEROL a

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PROTEINS

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90 80-

.~

7o

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6O

404 Z

30

~ 30 I0-

0

1;

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,'o

s'o POSTNATAL DAY

Fig. 6. Biochemical profiles of subcortical white matter during post-natal development in normal rats and in animals made hypothyroid at birth. The ordinate represents the amount of total lipids, cerebrosides, cholesterol, and proteolipid proteins expressed as percentage of the amount in normal specimens at 50 days. The post-natal age in days is shown on the abscissa.

As early as l0 days of age, the concentrations of cerebrosides in cortex and white matter were decreased in experimental animals. At 20 and 30 days, values in gray and white matter did not change compared to a 30 O//oincrease in age-matched controls. Between 30 and 40 days, cerebrosides normally double in both cortex and white matter. During this interval in hypothyroid animals, cerebrosides increase by only 33 °/o and 54 ~/o, yielding final concentrations in cortex and white matter which were 23"/ ;o a n a" 16 o~o, respectively, of normal values at 50 days. At 10 and 20 days of age, the concentrations of cholesterol in both cortex and white

166

N. H. BASS, E. YOUNG

matter of hypothyroid rats were decreased compared with controls. Between 20 and 30 days of age, cholesterol levels increased by 112°/~i in cortex and 154",; in white matter. This rise in the white matter of hypothyroid rats was similar to the increment shown in controls during the same period. Between 30 and 40 days, cholesterol norreally doubles in cortex and white matter. However, values in hypothyroid rats during this same period increased by only 65 ~o, finally attaining only 9" i in cortex and 13 ~o in white matter of control levels by 50 days. In the white matter of hypothyroid animals, the concentrations of proteolipid proteins were similar to control animals at 10 and 20 days of age. By 30 days, values were 33 ~ lower than controls. Between 30 and 50 days, levels increased less than two-fold in comparison with a four-fold increase normally found during this time. In the cerebral cortex of hypothyroid rats, the concentrations of proteolipid proteins at 10 and 20 days were increased significantly, but the rate of accumulation during this time paralleled that of controls. However, between 40 and 50 days, the conccntrations in gray matter of hypothyroid rats increased by only 18 °~ocompared to a 68 '!'i,rise in normal control specimens, resulting in values 33 %; below controls at 50 days.

DISCUSSION

Dwarfism in rats made hypothyroid at birth has been described in terms of the dramatic changes in their appearance (Goldberg and Chaikoff 1949; Scow and Simpson 1945). In addition to achieving only 40 ~'o of normal adult body weight, they exhibit infantile facial features, brachycephaly with reduced skull size, retardation in the development of teeth and a retention of short, soft and fine hair. Behaviorally, the time of eye opening is delayed (Eayrs and Lishman 1953) and the animals are lethargic, reflecting a decrease in their basal metabolic rate (Trojanova 1965; Trojanova and Mourek 1966). However, intermittent bursts of hyperactivity, frequently interrupting their usual sluggish behavior, are commonly observed. The rats huddle close to their mother or littermates and will follow a focal beam of light used as a heat source around the cage. This unusual behavior may be attributed to a permanent and irreversible defect of thermoregulation which has been previously reported in rats made hypothyroid at birth (Hamburgh 1968). The somatosensory cortex of the normal newborn rat contains poorly-differentiated neurons, closely-packed in vertical columns. The high cell density of the neonatal cortex is reflected by DNA values which are 20 ~o larger than those of the 50-day-old adult. Between birth and 10 days of age in both normal and hypothyroid rats, the evolution of the six-layered neocortex is accompanied by diminished cellular density which is reflected quantitatively by a more than 75 ~ decrease ofDNA levets. Since the ribosomes are concentrated in the perinuclear region of the cell, the concentration of RNA per unit volume is reduced also as the neuronal packing density decreases. The post-natal increase of gliaI cells in rat cerebrum has been well documented by morphologic (Caley and Maxwell 1968), radioautographic (Altman 1966), and microchemical studies (Bass, Netsky and Young 1969 a, b). Autoradiographic studies have also shown that in rats, cellular multiplication occurring postnatally near the ependymal surface of the lateral ventricles, is restricted largely to the production of

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glia (Adams and Davison 1965). From 10 to 50 days of normal postnatal development, the concentration of DNA increases progressively as glial cells migrate from the subependymal zone into cortex and white matter. The differentiation ofinterfascicular oligodendroglia is considered responsible for the myelination of axons in the central nervous system (Adams and Davison 1965 ; Peters and Vaughn 1970), which begins at 10 days and progressses most rapidly between 30 and 40 days. By the 50th day of normal development, the number of cells and the density of the intercellular matrix increase greatly as myelinated fibers and associated glia attain an adult appearance. Although neurons showed a normal packing density and were distributed in 6 layers in the cerebral cortex of hypothyroid animals at 50 days, decreased numbers of welldifferentiated glia were noted. Undifferentiated cells, destined to migrate into cortex and white matter, accumulated and remained near the ependymal surface and had a pyknotic appearance throughout postnatal development. These histologic observations suggested that in rats given ~31I at birth, the migration of glial cell precursors from the subependymal zone into the cerebral cortex was disturbed. In support of this hypothesis, biochemical data indicated a much more gradual increase of DNA, so that at 50 days, values in cortex and white matter were 49 ~ and 44 ~o lower than controls, respectively. Similarly, low values for RNA were found in cortex during this developmental period, further corroborating the theory of impaired migration ofglial cells and perhaps indicating some abnormality of the protein-synthesizing organelles of the neurons. In white matter of hypothyroid animals, however, an increase of RNA was found between 10 and 50 days, probably relating to an abortive attempt of undifferentiated glial cells to migrate through white matter from the subependymal layer. In the cerebrum of normal and hypothyroid rats, water content decreases by 15 o~, between birth and 50 days of age. At the same time, total solids normally double, the largest increment occurring between 30 and 40 days, when myelin lipids increase greatly (Eayrs 1961). Despite the initial decrease of dry weight per unit volume in the cerebrum of experimental animals during the first 10 days of life, a subsequent acceleration in the rate of accumulation of total solids was found, so that at 30 days, values approached those found in controls. However, between 30 and 50 days of age, total solids did not increase in the cerebrum of hypothyroid rats. Between 30 and 40 days when total lipid content normally doubles in developing animals, the concentrations of total lipids decreased strikingly, values in cortex and white matter being less than 50 of age-matched controls. The abnormal rate of accumulation of total lipid persisted between 40 and 50 post-natal days, resulting in concentrations in cortex and white matter which were 27 °/o and 40/o, o/ respectively, of 50-day-old control values. These changes inverted the normal ratio of proteins to lipids in the cerebrum of young adult hypothyroid rats, correlating with histologic observations of large decreases in stainable myelin. Previous microchemical and histologic studies have shown that in neural tissue~ the concentration of cerebrosides (including sulfatides) may be used as a quantitative index of the amount of myelin and, perhaps to a lesser extent, of oligodendroglia (Bass and Hess 1969; Bass et al. 1969 b; Fewster and Mead 1968). Although normally low in both cortex and white matter of 10-day-old rats, cerebroside concentrations were significantly decreased in hypothyroid animals. Between 10 and 30 postnatal

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days, cerebrosides increased only slightly in both cortex and white matter of hypothyroid rats, resulting in markedly lower values compared with age-matched controls~ This failure ofcerebroside accumulation in hypothyroid animals persisted throughoul development so that between 30 and 50 days, cerebroside concentrations in both cortex and white matter were less than 30 ~°~iof normal adult levels. These values reflect decreased formation of myelin associated with diminished proliferation of the plasma membranes of oligodendroglial cells and support previous findings showing that synthesis of sulfatides necessary for myelin formation is markedly decreased in the brains of hypothyroid rats (Walravens and Chase 1969). In addition to cerebrosides, cholesterol, which is generally regarded as a universal membrane constituent, may be used as a quantitative biochemical index of myelin. Both cholesterol and cerebrosides have been found to be greatly reduced in whole brain and purified myelin fractions from hypothyroid animals during the first 40 postnatal days (Balazs 1971 ; Balazs et al. 1969). In the present study, concentrations of cholesterol in cortex and white matter were decreased slightly in hypothyroid animals between 10 and 20 days. This was followed by a steady increase in concentrations so that at 30 days, values were 35 o~, lower in both cortex and white matter than age-matched controls. Between 30 and 40 days, the rate of cholesterol accumulation normally doubles ; in contrast, this component increased by only 6 ,,, in hypothyroid rats. Moreover, between 40 and 50 days, cholesterol concentrations in the cerebral cortex of hypothyroid animals decreased strikingly, so that levels were less than 20'!ii of normal values. Proteolipid proteins increase greatly during the most active phase of myelination m the white matter of developing rat cerebrum (Bass et al. 1969 b). However, comparatively large amounts of proteolipid in the presence of low cerebroside concentrations have been found in synaptosome fractions derived from normal adult brain (Lapetina, Soto and De Robertis 1968), in cerebral cortex during early phases of normal myelin ogenesis (Bass et al. 1969 b), and in poorly myelinated cortex resulting from undernutrition (Bass, Netsky and Young 1970). Therefore, in those areas of gray matter in the central nervous system where the amount of myelin is decreased normally or pathologically, proteolipid is a relatively poor index of myelin mass in comparison with cerebrosides. Between 10 and 40 days of age, the concentrations ofproteolipid proteins in the cerebral cortex of hypothyroid animals were equal to or greater than agematched control values, resulting in large increases in the ratios of proteolipid to cerebrosides. In the white matter of hypothyroid animals before 20 days of age, proteolipid concentrations were normal. However, between 20 and 50 days, when myelinogenesis usually proceeds at its most rapid rate and proteotipid proteins more than double, they increased by only 15 ° o. At 50 days, proteolipid values in the white matter of hypothyroid rats were only 45 i!, of age-matched control specimens. Therefore, although the concentration of proteolipid in the cortex was relatively increased as a function of its partial localization in neuronal membranes, the decreased accumulation of this myelin component in the white matter of hypothyroid rats could be correlated with defective myelinogenesis. The formation and maintenance of the myelin sheath depends upon structural and metabolic interrelationships between axons and interfascicular oligodendroglia. o/

~

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Furthermore, it has been suggested that the axon may act as an "organizer" in determining the onset of myelination (Friede 1961). Axons in subcortical white matter of the somatosensory area, as seen in silver impregnations and as measured by ganglioside assays, are not mature until at least 30 days of age (Bass et al. 1969 a, b). Since myelination depends on the maturation of axons as well as oligodendroglia, any metabolic insult interfering with the differentiation of neurons prior to 30 postnatal days, may result in decreased myelin. Total ganglioside sialic acid, a component of neuronal membranes, normally is highest in cortex and white matter at 20 post-natal days. This peak, attributed to disialoganglioside concentrations, is correlated with axodendritic proliferation and the formation of synaptic junctions, as well as with maturation of cortical electrical activity (Bass et al. 1969 a). By the 21st day, the development of adult food-seeking behavior normally allows weaning to take place. In contrast, animals made hypothyroid from birth, could not be weaned before the 35th day without producing a mortality of greater than 50 %. In these experimental animals, peak accumulation of ganglioside sialic acid occurred at 20 days but was diminished by 30 ~/o in both cortex and white matter. A similar decrease in ganglioside sialic acid was found in the cortex of the 50-day-old hypothyroid rats. This decrease was correlated with histologic observations of a lesser mass of neuronal fibers impregnated with silver. In the white matter of these animals, the ganglioside peak was also reduced, but the concentration of total sialic acid rose during subsequent development so that values at 50 days were slightly greater than control levels. These results suggest close relationships between the abnormal peak values in ganglioside sialic acid, decreased proliferation ofaxodendritic fibers and formation of synapses, and delayed behavioral maturation. Prior investigations in rats made hypothyroid at birth, have shown that the amplitude of the electroencephalogram recorded from the surface of the skull is markedly reduced and that neither an alerting nor a driving response to photic stimulation can be elicited (Bradley, Eayrs, Glass and Heath 1961; Bradley, Eayrs and Schmalbach 1960). Similarly, stimulation of the midline thalamic nuclei in conscious, unrestrained experimental animals produced a decreased amplitude, and increased latency and duration of negative-waves, as recorded from fhe cortical surface (Bradley, Eayrs and Richards 1964). A behavioral study of adult rats, hypothyroid since birth, showed a significant increase in the number of errors made during a fixed number of trials using the Hebb Williams closed field test (Eayrs 1961). Thus, it may be that the cerebral cortex in rats made hypothyroid at birth has a reduction of interneuronal connections and synapse formation which persists throughout postnatal life. Although this study revealed definite abnormalities in the rate of myelin formation associated with a defect in the migration and differentiation of glia, the pathologic contribution resulting from abnormally decreased axodendritic proliferation cannot be minimized. The present evidence suggests that a lack of thyroid hormone retards the maturation of cerebral cortex by inhibiting both the mitotic activity of glial cells and the differentiation of neurons and glia in the post-mitotic phase, thus resulting in a striking quantitative decrease in neural fiber mass. In the present experimental model, the contribution of at least two uncontrolled variables to the pathogenesis of the final cerebral lesion must be considered before

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attributing it solely to the effect of thyroid hormone deficiency. These include (1) neonatal undernutrition and (2) a diffuse disorder of endocrine imbalance merely intitiated by a lack of thyroid hormone. Since hypothyroid rats show not only delayed development of dentition, but a marked impairment in food-seeking behavior during the suckling period, a state of relative undernutrition may exist (Eayrs and Lishman 1953 ; Eayrs and Horn 1955). Moreover, it has been shown that a 21-day period of undernutrition begun immediately after birth is accompanied by chemical and histologic alterations in the somatosensory area of rat cerebrum, remarkably similar, although not identical, to those found in hypothyroid animals (Bass et al. 1970 a, b). Also, since the hypothalamic ~pituitary neurosecretory system of the albino rat is not functionally mature until at least 2 weeks after birth (Feldman, Vasquez and Kurtz 1961 ; Gorbman and Evans 1943), the production and release of hormones other than those secreted by the thyroid gland may be deranged in the hypothyroid animal. Indeed, other lesions of the endocrine system have been observed including degranulation ofacidophils and vacuotation of basophils in the pituitary gland (Scow 1959), as well as decreased weight and delayed maturation of gonadal and adrenal glands (Scow and Simpson 1945). Furthermore. manipulation of either adrenal, gonadal, or thyroid hormones during the perinatal period has been shown to alter the function of the other two systems (Levine and Mullins 1966). Therefore, it is difficult to interpret the results of the present experiment as a singular effect of decreased thryoid hormone. The changes observed in this and other similar studies of rats made hypothyroid at birth are probably only initiated by a lack of thyroid hormone, and are complicated and perpetuated by relative neonatal undernutrition, and a diffuse abnormality of endocrine secretion. The rat is a useful experimental animal for the study of developing cerebral cortex because of its extreme immaturity at birth and its rapid rate of post-natal growth. However, these very qualitites may preclude any exact correlations with human development. Thryoid function in the rat fetus does not begin until 2 to 3 days prior to birth, and synthesis of thyroid hormone does not reach adult levels until 2 to 3 weeks postnatally (Geloso et al. 1968; Samel 1968). In contrast, the human thyroid gland begins to function around the 16th week of gestation and reaches adult levels of hormone synthesis three months prior to birth (New England Journal of Medicine 1967). Moreover, the passage of thyroid hormones from mother to fetus across the placenta is extremely limited, so that maternal thyroid secretion cannot compensate for inadequate fetal hormonogenesis (Knobil and Josimovich 1958 ; Osorio and Myant 1962). Thus, rats made thyroid hormone deficient at birth may in some ways resemble human patients who are born without thyroid glands (athyrotic cretinism), in whom the pathologic effects on brain development are initiated, at least in part, during intrauterine life. In spite of severe behavioral retardation, only relatively minor pathologic changes have been detected in the brains of athyroid cretins by neuropathologic methods. It has been suggested that perhaps alterations of the cerebral cortex have occurred that are more quantitative than qualitative. The present study on the development of the cerebral cortex in the albino rat strongly supports this hypothesis, showing that hormonal imbalance initiated by hypothyroidism during post-natal life in the rat, and perhaps during perinatal life in man, may interfere with cortical maturation by retarding the differentiation of neurons and glka.

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SUMMARY

Microchemical techniques and histologic examination were used to study the somatosensory area of developing cerebral cortex and subcortical white matter in rats made thyroid hormone deficient immediately after birth. The body weight of these animals failed to show the normal growth spurt, achieving levels which were only 40 % of age-matched controls by 50 post-natal days. The endocrine imbalance initiated by a lack of thyroid hormone from the day of birth results in a metabolic derangement which disturbs the migration of glial cells from the subependymal zone into cortex between 10 and 50 days of post-natal life. During this same developmental period, the differentiation of neurons is impaired, resulting in decreased formation of axodendritic processes and synapses. Finally, in association with the pathologic development of axons and fewer numbers of well-differentiated interfascicular oligodendroglia, there is a failure of myelin formation. Hence, in rats made hypothyroid at birth, the cerebrum is subjected to drastic alterations in the normally continuous and interdependent process of neuron and glial cell differentiation.

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