THYROID DEFICIENCY AND INANITION :

THE EFFECTS OF REPLACEMENT THERAPY ON THE DEVELOPMENT

OF THE CEREBRAL CORTEX OF YOURG ALBIXO RATS

GABRIEL HORN Departnisnt of Anatomy, University of Birmingham, England

THREE FIGURES

The changes which occur in the central nervous system as a result of hypothyroidism vary according to the age of onset of the disorder. When thyroid deficiency first appears after the attainment of maturity, as in myxoedema, cerebral func- tion can usually be restored by treatment with thyroid hor- mone. On the other hand, the retarded mental development found in congenital hypothyroidism frequently fails to im- prove with replacement therapy.

Two hypotheses have been advanced to account for these observations. According to the first, thyroid hormone exerts a specific influence on the maturing cerebral cortex, and ab- sence of the hormone during a critical phase of development results in a disorganization of cerebral structure which cannot later be remedied (TVilkins, '38). Alternatively, it has been suggested that it is not thyroid deprivation so much as an associated, but undefined, congenital deficiency which may ac- count for the irreversibility of the cerebral changes common in cretinism (Bruch and McCune, '44 ; Tredgold and Tred- gold, '52).

Changes in cerebral structure have not been extensively studied in human cretins, but it has been found that hypo- thyroidism (produced early in post-natal life) in the rat is associated with : ( a ) retarded myelinization (Barrnett, '48), (b) a decrease in the size and a closer packing of the cortical neurones (Eayrs and Taylor, '51) and (c) a reduction in the

63

64 GABRIEL HORN

number of axons in the cerebral cortex (Eayrs and Horn, '55). Similar changes which do not appear to be due to the thyroid deficiency (Eayrs and Horn, '55) follow chronic inanition during infancy (Schukow, 1895; Sugita, '18). I n the present experiment use has been made of this similarity in a n at- tempt to account for the apparent irreversibility of cerebral dysfunction in the cretin. The effects of inanition are pre- sumably non-specific, and the view that thyroid hormone ex- erts a specific influence on the growth of the cerebral cortex has accordingly been examined by comparing the results of replacement therapy on the cortical structure of both cretinous and starved animals.

MATERIALS AND METHODS

Seventy-two newborn albino rats of the "Birmingham" strain maintained at this laboratory were used. They were fed on Thomson rat diet and given water ad l ib. Certain groups of animals (see below) were starved according to the method described by Eayrs and Horn ( '54). Thyroid activity was suppressed by a single subcutaneous injection of 100 pc of carrier-free radio-active iodine (Goldberg and Chaikoff, '49).

Experimental design

In order to extract as much information as possible from the observations, a balanced incomplete block design was used and the treatments arranged accordingly (Cochran and Cox, '50, plan no. 11.11). The animals were arranged in three groups, each containing 24 rats (see table 1). All animals in groups I1 and 111 received 1131 on the day of birth; those in group I did not. The animals of group I1 were given thyroid replacement therapy in the form of daily subcutaneous injections of 2 v g of 1-thyroxine sodium, from the 24th day after birth until death.

The three main groups of 24 animals each were subdivided into three cells, each of which contained 8 rats (see table 1). Animals in cell 1 were given a full diet throughout the experi-

THYROID DEFICIENCY AND INANITION 65

mental period ; those in cell 2 were underfed from birth to the 24th day, after which they were allowed a full diet until death; the animals in cell 3 were underfed from bith until they were killed. All animals were killed on the 60th day after birth.

In accordance with the design of the experiment, 18 litters were used, each of which contributed 4 males to the experi- ment. One additional male litter-mate was kept to replace the one receiving the most severe treatment, in case it should die. In those litters in which three or 4 animals had to be removed for a daily period of starvation, one or two female litter-mates were left with the mother, thereby ensuring a constant stimulus to lactation.

TABLE 1

Arrangement of treatments

GROUP 111 GRQUP I1 GROUP I No 10’ In1 +Thyrox ine from 24th day 1131

Cell I Cell I1 Cell 111 Cell I Cell I1 Cell I11 Cell I Cell 11 Cell 111

No Partly Under- No Partly Under- No Partly Under under- under- fed under- under- fed under- under- fed feed- fed feed- fed feed- fed ing ing ing

(0-24 (0-60 (0-24 (0-60 (0-24 (0-60 days) days) days) days) days) days)

Owing to the fact that cretinous and starved cretinous rats were unable to feed adequately on the standard diet, weaning was often delayed beyond the 28th day in litters containing such animals.

Autopsy and histological methods

Each animal was weighed immediately before being killed with chloroform. The brain was rapidly removed, placed in a known quantity of chloral hydrate and weighed. The thy- roid glands of all the animals in group I were dissected, weighed and fixed in Bouin’s solution. The larynx, upper part of the trachea and attached structures were removed from animals of groups I1 and I11 and fixed in Rouin’s solution.

66 GABRIEL HORN

The thyroid glands from animals in group I, and the laryngeal region of other animals, were serially sectioned longitudinally at 5 p and stained by Koneff's modification of Mallory's azan method (Tala, '52). The brains were sec- tioned in the coronal plane along a line immediately anterior to the mammillary bodies. Several sections in this plane were cut at 10 p, mounted and impregnated with silver by the tech- nique of Nonidez ( '39) and counter-stained with methylene blue to stain the nerve cell bodies.

Collection of duta, ( a ) Thyroid activity. Tala ('52) compared several quan-

titative methods of assessing thyroid activity and concluded that this could best be done by estimating the amount of alveolar epithelium present. I n the present study, the pro- portion of thyroid tissue occupied by epithelium, colloid and interstitial substance was estimated by Chalkley's ( '47) method. The percentage of epithelium present was considered to represent the functional state of the gland, and was de- termined from 10 regularly spaced sections through each lobe, a total of 20 sections per gland. Serial sections of the larynx and trachea from all animals which received II3l

were examined for the presence of thyroid tissue. ( b ) Fibre and cell counts. Fibre and cell counts were

made in the laminae pyramidalis and ganglionaris in the an- terior par t of area striata (Krieg, '46). The lamina gang- lionaris was recognised by the presence in it of large ganglion cells. Lamina pyramidalis was most readily identified as lying between layers I1 and IV, both of which could be easily recognised. Fibre and cell counts were made in 5 adjacent fields in each layer respectively. The methods used for connt- ing fibres and cells were similar to those previously described (Eayrs and Taylor, '51; Eayrs and Horn, '55).

RESULTS

Only 43 of the 72 experimental animals lived to the end of the experiment. h total of 19 animals in group I survived,

THYHOID DEFICIENCY AND INARITION 67

14 in group 11 and 11 in group 111. There was only one sur- vivor in cell I11 of group I11 so data from this animal were not included in the analysis. As a result of the high mortality rate, the factorial design broke down. Because heterogeneity of variance was often observed between the treatments, a ranking test (White, '52) was used to test comparisons be- tween different treatments. A probability level of 0.02 or less was adopted as the basis for testing for statistical significance.

Thyroid tissue S o thyroid tissue could be found on microscopical ex-

amination in 18 of the 24 surviving 1131 treated animals. I n the remaining 6 rats, a small quantity of atypical glandular tissue was present. The cytoplasm of many of the cells of this tissue was vacuolated and showed diminished granula- tion. The nuclei were large and pale and the intra-alveolar colloid, where present, was less strongly acidophilic than the colloid in normal glands. I t seems probable that the func- tional activity of the surviving thyroid tissue in these animals was severely depressed.

The thyroids of the partly underfed and of the underfed animals in group I were removed with the aid of a dissecting microscope. These glands were so small and translucent that it was impossible to separate the connective tissue satisfac- torily from them, and they were therefore not weighed. The percentage of thyroid tissue occupied by acinar epithelium in normal, underfed and partly underfed animals was 65.20

4.72, 65.17 r+ 3.61, and 66.79 t 4.53 respectively. There was no significant difference between these values (P > 0.2). No structural differences were found between the glands of the normal and experimental animals. It is concluded that the fmictional activity of the thyroid is not depressed in chronically underfed and partly underfed rats.

Growth 1. Brain amd body weights. Data of brain and body

weights are given in tables 2 and 3. These show that diff-

68 GABRIEL HORN

ercnces in body weight between experimental and control animals are associated with corresponding differences in brain weights. Thus both body and brain weights of the cretinous and of the underfed animals are significantly smaller than the corresponding weights of the matched controls. Generally speaking, the brain and body weights of the thyroxine-treated and of the partly underfed rats do not differ significantly from the corresponding weights of the control animals (see tables 2 and 3) .

TABLE 2

Comparison of body weights between experimental and control rats

MEAN BODY PROBA- NUMBER WEIGHT BILITY

ANIMALS O F MEANS D I F P E B FACTOR TREATMENT OP STANDARD ERROR OF

( G M ) ENCE

Thyroid deficiency

{Noe;under- ( Control

[ye;under- [Control

7 206.50 t 31.06 < 0.01

24 days (Thyroid deficient 5 51.80 t 15.93 < "01-

{ Thyroid replace- 6 142.67 t 21.73 < 0'01

Thyroid deficient 5 49.60 ? 10.83 Underfed to Control 8 138.19 t 16.39

~~

7 206.50 t 31.06

c ment therapy Thyroid replace-

therapy

8 138.19 t 16.39 N.S. replace- 5 108.80 C 26.56

ment therapy 4 43.875 t 6.813

N.S. replace- 3 37.67 t 4.935 ment therapy

Under- intact Underfed to 60 days 4 43.875 -C 6.813 7 206.50 t 31.06 < 0.001

-~ ~

Thyroid Control

feeding [TI:. { (0-60 days) Underfed to 60 days 3 37.67 C 4.935 < O.Ool

Thyroid Control 7 206.50 t 31.06 138.19 ? 16.39

feeding replace- Control 6 142.67 C 21.73

6 142.67 t 21.73 replace- Control

therapy

merit i __

I Underfed to 24 days 8 < O.Ool Partial under- Thyroid ' intact [Vnderfed t o 24 days 5 108.80 t 26.56 N.S*

5 49.60 C 10.83 to 24 days 5 51.80 C 15.93 N.S'

N.S. = Not significant.

THYROID DEFICIENCY AND INANITION 69

Thyroid replace- ment therapy

The brain weights of the 24-day-old aiiimals, both starved and hypothyroid (Eayrs and Horn, '55) were compared with the brain weights of similarly treated animals in the present experiment. There is no significant difference in weight be- tween the brains of the 24 and 60-day-old cretins. This re- sult indicates that no obvious growth takes place in the brains of hypothyroid rats after the 24th day. On the other hand to

8 1.5003 C 0.07228 Thyioid replace 5 1.3857 It 0.2138 N.S.

Underfed (Control

inent therapy 4 1.2549 2 0.07134

to 24 days

Underfed Control

1 therapy ( 5 1.3136 & 0.1119

to 24 days 5 1.2087 :k 0.1384 N.S* Thyroid I days)

X.S. = Not significant.

70 GABRIEL HORN

keep the mortality as low as possible, the underfed animals were allowed to grow daily throughout the experiment. As a consequence the brains of the 60-day-old underfed rats weigh 1.25 0.07 gm while the brains of the vounger animals weigh 0.93 0.10 gm. The difference is significant (P <

When the data for laminae 111 and V were compared between experimental and control animals,

0.001). 2. Cerebral cortex.

TABLE 4

Comparison of combined fibre counts (layer III + layer V ) between experimental and control rats

FACTOR TREATMENT

PROBA- BILITY

OF DIFFER-

ENCE

NUMBER MEAN FIBRE COUNT

STANDARD ERROR O F MEANS

Thyroid deficiency

Thyroid replace- ment therapy

Kot under- Control fed {Thyroid deficient

to 24 days I Thyroid deficient

Thyroid replace- ment therapy

fed

r ment therapy

Underfed Control

IJnderfed Control t o 24 days Thyroid replace-

Underfed (Control to 60 days 3 Thyroid replace-

7 237.57 t 20.17 5 151.80 i 22.52 < 0'01 8 243.88 t 25.26 5 145.00 t 16.64 < 0'01 7 237.57 2 20.17 6 202.83 t 22.65 < 0'05

8 243.88 i 25.26 5 191.80 t 33.17 < 0'05

4 234.00 i 35.86 3 163.33 zk 27.51 < 0'05

nient therapy

Control 7 237.57 t 20.17 Under- Underfed t o 60 days 4 234.00 ? 35.86

Control 6 202.83 1 2 2 . 6 5 Underfed to 60 days 3 163.33 t 27.51

Thyroid Control 7 237.57 & 20.17 Underfed to 24 days 8 243.88 2 25.26

6 202.83 t 22.65 Underfed t o 24 days 5 191.80 i 33.17

5 151.80 t 22.52

N.S.

N.S.

~~

therapy

N.S.

N.S. nnder-

N.S. t o 24 days 5 145.00 t 16.64

N.S. = Not significant

THYROID DEFICIENCY AND INANITION 71

it was found that the significance of the differences remained the same whether the layers were compared separately, or the data added together (laminae I11 + V) and then compared. As a result, only data for the combined values are given. The one exception to these findings is mentioned below.

Data given in tables 4 and 5 indicate that the development of the cerebral cortex of cretinous rats is severely retarded.

TABLE 5

Comparison o f .combined cell counts ( layer 111 + layer 8) between emperimental and control ra ts

FACTOR

PROBA- BILITY

O F DIFFER- ENCE

NUMBER MEAN CELL

A , ~ ~ A , s STANDARD ERROR COUNT

O F MEANS

TREATMENTS

Thyroid deficiency

~

-Not under- Control 7 70.43 rt 5.75 fed Thyroid deficient 5 93.80 2 11.06 < O.O1

. to 24 days Thyroid deficient 5 97.20 f 9.10 < O.O1 Underfed {Control 8 76.75 rt 5.49

Thyroid replace- ment therapy

Control 7 70.43 f 5.75 Thyroid replaoe- 6 71.17 2 3.19 N.S. "Ot fed under- 4 ment therapy

Underfed Control 8 76.75 rt 5.49 to 24 days {Thyroid replace- 5 77.00 2 16.98 N.S.

I ment therapy 4 84.00 zk 6.68

replace- 3 97.00 f 11.53 N.S. ment therapy

Thyroid Control 7 70.43 rt 5.75 {Underfed t o 60 days 4 84.00 rt 6.68

Control 6 71.17 & 3.19 Underfed to 60 days 3 97.00 f 11.53 < 0.05

intact < 0.05

therapy

Under-

Thyroid Control 7 70.43 rt 5.75 intact {Underfed to 24 days 8 76.75 & 5.49 N.S.

6 71.17 & 3.19 Underfed to 24 days 5 77.00 & 16.98

Control 5 93.80 & 11.06

N.S. under-

N.S. to 24 days 5 97.20 rt 9.10

N.S. = Not significant.

72 GABRIEL HORN

Area striata of the brains of these animals contain signifi- cantly (P < 0.01) fewer fibres and significantly (P < 0.01) more cells than those of the controls. Lamina ganglionaris of the cretinous rat brain does not, however, contain signifi- cantly more cells than the corresponding layer of the controls. T ~ L I S tlie mean values of the cell counts in the hypothyroid animals are 33.60 -+ 3.20 and 40.68 I 8.41, whilst the cor- responding values in the matched controls are 30.29 i- 3.98 and 33.38 rtl 5.63 respectively.

The cortex of the cretinous rats which received thyroid medication from tlie 24th day after birth, has been largely restored to normal. Table 5 shows that the combined cell counts of these animals are not significantly different from their controls. The combined fibre counts of the animals treated with thyroxine (table 4) are smaller, though not sig- nificantly so (P < 0.05 in each case) than those of the con- trols. The higher mean cell counts of the starved animals are not Significantly different (P < 0.05 in each case) from the mean counts in the corresponding controls. The structure of the cortex of the starved animals re-fed from the 24th day after birth is similar to that of the matched controls.

A linwr relation exists between brain weights and com- bined cell counts (fig. 1). The correlation coefficient (-0.78) is significantly different from zero (P < 0.001). Thus there are more cells per unit area of cortex in a small brain than in a heavier brain. This inverse relationship between brain weight and cell density suggests that the total number of cells in the cortex of a small brain may not differ greatly from the total number of cells in the cortex of a heavier brain. On the other hand, a highly significant correlation (P < 0.001) between brain weight and fibre count (fig. 2) indicates that brain weight is directly related to fibre density. These re- sults suggest that differences in brain weight are associated with differences in fibre density, rather than with differences in the total number of cells in the cortex.

Combined cell counts are plotted against combined fibre counts in figure 3. The correlation coefficient (-0.60) is sig-

. ( 1 0

280-

v) t Z 3

w z 2 150

0 0

g 2 0 0

0

0 0 - 0

0 0 0

0

O o o / - 0 9 0 D O

;.----/Too 0

0 0

0

U 0

0

0 0 0

-I - o _I w - u 7 5 - 0

2 - f -

F m - I . 0 0 5 0 ' l l l a * ~ ~ * ~ * * * ~ I ,

0

0 8

120 150 2 0 0 2 5 0 COMBINED FIBRE COUNTS

Fig. 3 The correlation coefficient = -0.60 and is significantly different from zero (P < 0.001).

Line E = Regression line of combined fibre counts on combined cell counts. Line F = Regression line of combined cell counts on combined fibre counts.

The relationship between combined fibre counts and combined cell counts.

73

74 GABRIEL HORN

nificantly different from zero (P < 0.001). Thus a high cell density is associated with a low fibre density.

DISCUSSION

No quantitative differences have been observed between the functioilal activity or appearance of the thyroid glands of underfed and normal animals. This suggests that the effects of inanition on the developing cortex of the young rat are not Secondary to a depression of thyroid activity.

The figures also suggest that the total number of neurones in the cerebral cortex may be constant in all the animals ir- respective of treatment, whereas the density of the fibre network varies with the brain weight. In general it was found that a high cell concentration is associated with a low fibre density and vice versa. A similar relationship between fibre and cell density is found in the brains of young starved and young cretinous rats (Sugita, '18 ; Eayrs and Taylor, '51 ; Eayrs and Horn, '54). Thus the adverse effects on brain weight observed in these experiments seem to be secondary to a change in fibre density, rather than to any change in cell numbers. If the density of the fibres in the cortex is regarded as an index of cortical development (Conel, '41, '47), then the treatments have effected differentiation rather than mul- tiplicative growth of the cortex. These general conclusions are not, however, adequate to account for the conditions found in 60-day-old starved rats, for in these animals the brains weigh significantly less than those of the controls, have a slightly higher concentration of cells but have a fibre network of equal density. The following hypothesis is advanced to account f o r these differences. Allen ('12) has shown that the cerebral cortex of the rat has nearly its full complement of cells by the 20th day after birth. The low concentration of fibres in the cortex of the 24-day-old starved rat (Eayrs and Horn, '54) and the high concentration of cells in rats of comparable age (Sugita, '1 8) indicate that underfeeding retards differential growth but does not effect multiplicative growth. Up to the 24th day therefore, much of the available

THYROID DEFICIENCY AND INANITION 75

protein is probably appropriated by the rapidly forming, but immature, cortical cells. These cells may be immature because, as Sugita ('18) has shown, they are smaller than those in normal control animals. A small cell has a higher surface area/volume ratio than a larger cell. Therefore a small cell is a more efficient system than a large cell in conditions of undernutrition, since it exposes a relatively large surface to the nutrient fluid. Hence the optimum cell size is reached at an early stage in development and the cell divides in a rela- tively immature state. After the 20th day multiplicative growth has practically ceased and, provided the animal is given an adequate amount of protein, this will be used by the cells for differential growth.

The effects of thyroid deficiency on the growth of the cortex does not appear to be materially altered between the 24th and 60th day. The brain weights at the two ages are not significantly different. At each age the cretinous brains weigh less than those of the normal controls, have a higher cell concentration and a lower fibre count. Within the time limits of the experiment, thyroid deficiency, in contrast to dietary starvation, appears to retard cortical maturation permanently.

It is conceivable that even when the intake of food is suffi- cient, a lack of thyroid hormone may prevent the cells from absorbing various substances transported in the blood stream essential to their normal growth and function. When multi- plicative growth has ceased the nerve cell would be unable to use the additional and available portein for the development of its protoplasmic processes. The cause of the dysfunction may be either intra-cellular or extra-cellular. I t is unlikely that the intra-cellular enzyme systems are affected in thyroid deficiency, for the &a vitro oxygen consumption of the develop- ing cortex of cretinous rats is the same as that in normal controls (Fazekas, Graves and Alman, 51). Provided there- fore that the enzyme systems are intact, the amount of nu- trients used will depend on the amount presented to the cell. This, in turn, will depend on ( a ) the source of supply (in the blood) and (b) the ability of the nutrients to pass from

76 GABRIEL HORN

the capillaries to the cell. Concerning the first of these fac- tors there is no evidence that hypoglycaemia or hypoprotein- aemia exists in hypothyroidism (Deusch, ’20 ; Benda, ’47). Furthermore, it is unlikely that the brain is subjected to an anoxaemia, for Himwich, Daly, Fazekas and Herrlich ( ’42) have shown that in human cretins the difference in cerebral arterio-venous oxygen is 5.74 volumes ”/., while that for normal adults is 7 volumes %. Himwich et al. consider that this difference between cretinous and normal humans may be even greater because of the slow blood flow through the brains of the cretins. These results indicate that the cretinous brain receives an adequate supply of oxygen, but uses less than the brain of normal controls. Alternatively, inanition may be brought about by interfering with the passage of nutrients from the capillaries to the cell, as from a change in the struc- ture of the cell menibrane (Hoffman and Hoffman, ’44) or of the ground substance which surrounds ( HPSS, ’53) the cells of the cortex. In this connection it may be significant that changes in the viscosity of the ground substance are asso- ciated with growth (Gersh and Catchpole, ’49).

Thyroid replacement therapy instituted on the 24th day af- ter birth is largely successful in restoring the organization of the cretinous cortex to normal. These results may account for the findings that replacement therapy leads to improved maze performance in young rats (Eayrs and Thhman, ’54). The position of human cretins receiving thyroid medication however, is somewhat different. Brown, Rronstein and Kraines (’39) found that the mental development of cretins receiving replacement therapy may remain severely retarded. Tredgold and Tredgold (’52)’ on the other hand, consider that provided treatment is commenced during the first year of life, many cases achieve a “normal mental standard.” These differences in response to thyroid medication have led Tredgold and Tredgolcl to suggest that there may be two types of cretins. In one typt. the physical and mental retardation may be due to hypothyroidism alone, and such cases respond well to treatment. I n the other type, mental retardation and

THYROID DEFICIENCY AND INANITION 77

thyroid hypoplasia may both be due to a co-existent congenital deficiency and in these cases mental development will not be stimulated by thyroid medication. The reversibility by re- placement therapy of the retarded cortical development in cretinous rats suggests that any failure to respond to ade- quate amounts of thyroxine may in fact be due to some eo- existent congenital deficiency.

SUMMARY

1. Studies were made of the effects of inanition and thyroid deficiency on the structure of the cerebral cortex of young rats. Starvation was brought about, starting on the second day after birth, by separating animals from their mothers for several hours per day. Thyroid activity was sup- pressed with P1 administered on the day of birth.

Commencing on the 24th post-natal day, some of the cretinous animals received thyroxine replacement therapy and some of the underfed animals were allowed to feed ad lib until the end of tlie experiment. All animals were killed on the 60th day after birth.

The brains of the thyroid deficicnt and chroiiically un- derfed rats weighed significantly less than the brains of their respective controls.

4. Fibre and cell counts were made in one area of the cerebral cortex, area striata. There were more cells and fewer fibres in this area in the hypothyroid than in the control ani- mals. The fibre and cell concentrations in the partly under- fed, chronically underfed and thyroxine-treated animals re- spectively did not differ significantly from tlie concentrations in the corresponding controls.

The total number of nerve cell bodies in the striate area of the cerebral cortex was constant in all the animals, ir- respective of treatment, whereas the density of the fibre net- work varied with brain weight. In general, a high cell con- centration x7as associated with a low fibre density and c i c e versa.

2.

3.

5.

78 GABRIEL HORN

6. Quantitative estimation of thyroid function did not dis- close any significant difference between the activity or ap- pearance of the thyroid glands of normal, partly underfed and chronically underfed animals.

These findings are discussed, and it is suggested that lack of thyroid hormone may prevent the neurones from ab- sorbing various substances transported in the blood stream, essential to their normal growth and function, so that beyond a certain stage, development of the neuropil is prevented.

7.

ACKNOWLEDGMENTS

It is a pleasure to thank my wife, Ann Horn, B.Sc. for as- sessing the activity of the thyroid glands, Dr. Deryk Darling- ton for his advice on the statistical treatment of data and Professor S. Zuckerman for reading the typescript and offer- ing many helpful suggestions.

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THYROID DEFICIENCY AND INANITION 79

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