23, 51-57 (1969)
C. DIAMOND, RUTH E. JOHNSON, CAROL INGHAM, AND BEATRICE STONE 1
of Physiology-Anatomy, Berkeley, California Received
An investigation was carried out to determine the effects of early hypophysectomy (5-6 days of age) and later hypophysectomy (28 days of age) on brain morphology. Graded doses of growth hormone were injected into normal postnatal rats to compare the brain anatomy with that from the early hypophysectomized rats. The animals were divided into five groups as follows. Group I: The rats were hypophysectomized at 5 days of age and killed at 26 days of age. Replicable somatosensory cortical depth differences were not obtained, possibly due to variarion in hypophysectomy techniques. The occipital cortex was not affected by hypophysectomy in these series of experiments. Group II: Littermate rats were hypophysectomized at 6 days of age and killed at 41 days of age. The somatosensory cortex of the hypophysectomized rat was significantly deeper than the control, the difference being attributed to altered skull development. Group III: Rats were hypophysectomized at 28 days of age and killed at 77 days of age. No brain changes were noted, though marked body weight differences were evident. Group IV: Twenty-one micrograms of growth hormone were injected into normal rats in graded weekly doses beginning at 7 days of age and continuing for 3 weeks. No brain changes or body weight differences were found when compared with uninjected controls. Group V: A total of 300 fig was divided into graded, daily injections, beginning at 7 days of age and continuing for 3 weeks. No brain changes or body weight differences were seen when compared with uninjetted controls. Introduction The relationship between the development of the nervous system and the endocrine system has been studied from numerous approaches (5, 8, 10, 13). Our investigations have been primarily concerned with the effects of early hypophysectomy on brain growth. We have previously reported the following observations after early hypophysectomy in the postnatal rat compared with normal control littermates: (a) No differences in brain weights were noted in either whole brains or in dissected parts (6, 7). 1 This work was supported by USPHS grant NB 06293-02. The authors wish to acknowledge the technical assistance of Mrs. Jacqueline Ehlert. 51
(b) No differences were measured in cerebral cortical depth (4), or in the branching of basal dendrites of pyramidal cells in layers II and III of the somatosensory cortex (6). (c) The diencephalon dimensions were altered, with the depth of the diencephalon being more consistently increased than the width (2 j. (d) Thyroxine increased cortical growth whereas growth hormone did not (2). (e) No differences were found in cerebral cortical protein (7). (f) Acetylcholinesterase activity per utiit weight decreased in the somatosensory and ventral cortex, and cholinesterase activity per unit weight decreased in the hypothalamus. In other brain areas sampled, these two enzymes did not differ between the hypophysectomized and normal rat (7). (g) Marked differences were evident in body weights (2,6,7) and endocrine organ weights (6,7). In the present experiments, several of the previous conditions were varied further in order to determine if morphological cortical changes could be brought about through hypophysectomy and with growth hormone administrtiion to normal animals. Some animals were hypophysectomized 1 day earlier than in the previous experiments: at 5 clays of age instead of 6 (Group I). Some rats were allowed to live for longer periods of time without their pituitaries ; for 5 weeks instead of 3 (Group II). Some had their pituitaries removed at 28 days of age instead of 6 clays of age (Group III). Growth hormone was administered in varying doses to normal animals to compare their brains with those from hypophysectomizecl animals injected with growth hormone (Groups IV ancl V) . Methods
Male, Long-Evans rats were used throughout these experiments. Detailed descriptions of the surgical procedures, the histological techniques for frozen sections and the brain measurements have been published (2, 3, 4). However, a brief description of the histological techniques is as follows: At the termination of each experimental condition, the rats were anesthetized with ether and perfused with 10% formol-saline solution through the left ventricle of the heart. The brains were removed from the skull, examined for hypothalamic damage, and placed in 10% formol-saline solution. All skulls were examined for pituitary remnants in the hypophysectomized rats. With the exception of the Group I experiment, frozen sections were taken. Transverse sections were cut at 20~~ utilizing specific subcortical landmarks to insure uniform sampling. The anterior commissure was used as a reference point to designate the overlying somatosensory area, and the posterior commissure served as the landmark for the overlying occipital cortex. In the Group I experiment, the brains were embedded by two different celloidin techniques. For the Group I-A, a slow method required 1 week
through the graded alcohol treatment and 8 weeks from 10% celloidin through 50% celloidin. For the Group I-B, a rapid celloidin procedure was followed, namely 36 hours in the graded alcohols and 36 hours in celloidin in a 56” oven. For both methods the celloidin was hardened slowly, 2-4 weeks. The celloidin sections were cut at 10~ from regions identical to the frozen sections. Thionine stain was used throughout these experiments (12). Cortical depth and diencephalon measures were taken on all tissues in a manner reported in detail previously (2). Briefly, the cortical and subcortical outlines were projected with the microslide pro jector (enlarged X22.5) and drawn. Beginning lateral to the elevation on the corpus callosum, five cortical depths were read with a millimeter rule extending from the white matter to the pial surface. Both right and left hemispheres were read, and the results are presented as the means of both hemispheres. For the diencephalon measures, two linear readings with a millimeter rule were taken: A vertical midline depth reading extending from the dorsal surface to the ventral surface of the diencephalon and a horizontal measure between two points demarcating the widest lateral distance in the diencephalon. The individual experimental conditions are presented in the following groups : Group I. A. Ten nonlittermate pairs of rat brain were studied with one rat in each pair hypophysectomized at 5 days of age. The rats were killed at 26 days of age. The slow celloidin embedding technique was used. B. One rat in each of ten nonlittermate pairs was hypophysectomized at 5 days of age, and the pairs were autopsied at 26 days of age. The rapid celloidin embedding technique was followed for these brains. Group II. One rat from each of 13 littermate pairs was hypophysectomized at 6 days of age; all 13 pairs were killed at 41 days of age. Group III. One rat in each 19 littermate pairs was hypophysectomized at 28 days of age ; all animals were killed at 77 days of age. Group IV. A total of 21 pg of growth hormone (9) was administered subcutaneously to 20 normal rats beginning at 7 days of age and continuing for 3 weeks. The hormone was divided into three single, weekly graded doses: 3.5, 7, and 10.5 pg. The dosage was comparable to that given to rats hypophysectomized at 6 days of age (2). For every injected rat there was an uninjected littermate control. Group Y. A total of 300 pg of growth hormone (9) was administered to 20 normal rats beginning at 7 days of age and continuing for 20 days. Subcutaneous daily injections were given in amounts of 10 pg per day the first week, 15 pg per day the second week, and 20 pg per day the third week. The brains from the 20 growth hormone treated animals were compared with 15 uninjected littermates.
ET AL. Discussion
Results of all the experiments were tabulated. Copies of the tables will be furnished to readers upon request. Group I. The survival rate of rats hypophysectomized at 5 days of age is extremely
rats, only 20 survived
26 days. Unfortunately, these are the very brains on which conflicting results occur. In Group I-A brains, the depth of the somatosensoryarea of the hypophysectomized rats was less than the control by 12% (P < .OOl) ; in the replication experiment, Group I-B, the differences were not significant. In contrast, the occipital cortex showed similar nonsignificant differencesin both the initial and replication experiments. In three experiments on 6-day hypophysectomized rats, no cortical depth changeshave been noted in the somatosensory area. It is possible that the technique of hypophysectomy changed after the Group I-A experiments, for these were the first animals that we hypophysectomized at 5 days of age. Even though different celloidin embedding methods were used in preparing the tissues for this experiment, we do not believe this to be a causative factor for the cortical differences between experiments A and B, Group I. When comparing frozen sections with celloidin sections, cortical depth differences between the initial and replication experiments have been found to be similar regardlessof technique (3). The diencephalon depth increased in the hypophysectomized rat, whereas the diencephalon width did not. This finding will be discussedin conjunction with the following Group. Group II. As we have reported previously, when the pituitary was removed at 6 days of age and the rat was killed 21 days later, there were no measureable changes in the cortical depth in comparison with the brains from normal, intact rats (2). From this finding, the hypothesis was formulated that enough thyroxine was probably circulating through the brain to maintain normal, cortical development. However, Rosenberg (personal communication) found that 2 weeks after hypophysectomy of the 28-dayold rat, the circulating protein-bound iodine had decreasedby a factor of two and that 8 weeks after hypophysectomy, circulating protein-bound iodine had decreasedby a factor of four. From this information, one could speculate that morphological decreasesmight be expected 5 weeks after hypophysectomy at 6 days of age due to a diminished thyroxine level. Contrary to finding, the hypothesized decrease in cortical depth for the S-week period, the somatosensory area of the hypophysectomized animal was actually deeper than the control by 5.6% (p < .Ol). There were no significant differences in the occipital cortex. In the diencephtlon measures at the anterior commissure level, the hypophysectomized animal’s brain was deeper than the control by 5.2%
‘(p < .Ol), but the width differences were not significant. At the posterior commissure level, the hypophysectomized animal’s diencephalon was markedly greater in depth than the control by 13% (p < .OOl). Though the difference was not as great, the width of the diencephalon at this level was also significantly greater in the hypophysectomized animal than in the control (3.4%, p < .OOl). Thus, in four out of six measures, two cortical and four diencephalic, the hypophysectomized brain was greater than the control in spite of the marked body weight differences in the opposite direction. That the depth of the diencephalon was greater in the hypophysectomized animal at 41 days was not surprising, for already at 27 days this depth increase was evident (2). Walker et al. ( 11) had shown that the length of the cranium prematurely ceased to grow in the early hypophysectomized animal and that a “bulge” occurred in the roof of the cranium. This change in shape might account in part for the increase in the diencephalon depth. They also reported that the width of the cranium was increased by hypophysectomy. Our 27 and 41-day brain measures were discrepant here, for at 27 days there was a significant width increase only in the anterior commissure level, and at 41 days a significant width increase was noted only at the posterior commissure level. Why this shift occurs is not clear. The brain dimensions of the young rat in general, however, appeared to be governed more by limitations or the direction of skull growth, or both, rather than by direct action of hormone deficiencies on the brain. Group III. For most endocrine studies in our laboratories, rats are hypophysectomized at 28 days of age. Therefore, this age was chosen to study the effect of later hypophysectomy in contrast to early hypophysectomy on brain development. The body weights of the hypophysectomized animals plateaued 1 week postoperatively and at autopsy (77 days) weighed only a quarter as much as the control littermates. In spite of the stunting of body growth, there were no significant changes in the cortical depth measurements or in the diencephalon measures when compared with intact controls. The effects of this treatment on skull and on brain can be compared. Asling and Frank ( 1) demonstrated skull changes in the rat at 80 days of age as a consequence of hypophysectomy at 28 days of age. They reported that the width and length of the control rats’ crania were greater than those of the hypophysectomized rats, whereas there were no differences in cranial heights between the two groups. We have found no brain dimension changes in rats hypophysectomized at 28 days and autopsied at 77 days. These findings represent a case where brain dimensions in the older rat as measured by us and skull dimensions as measured by Asling, do not necessarily accompany one another. Groups IV and V. As mentioned in a previous report (2)) 21 pg of
growth hormone injected into hypophysectomized rats stimulated the body weight so that in three weeks it became 19% (p < .05) greater than that of the uninjected, hypophysectomized rat. The somatosensory and occipital cortices from these two groups of rats did not differ in depth measurements, but the diencephalic widths were significantly greater in the hypophysectomized, growth hormone treated animals. In the present experiment, we have given the identical dosage to normal animals, and no significant differences were noted in the body weights, or in cortical depth measurements, or in the diencephalon measures. A larger dosage, 300 pg, administered daily to normal intact rats from 7 to 28 days of age also yielded no significant differences in brain measures or in body weight. Thus, neither dosage of growth hormone administered to normal animals had an effect on body weight or brain measures, but 21 pg of growth hormone in the early hypophysectomized animal increased both the body weight and the width of the diencephalon. We were interested in administering growth hormone to young, intact rats to observe brain changes even though we were aware that body weights might not be altered. Zamenhof, Mosley, and Schuller (13) found that injecting purified bovine pituitary growth hormone into pregnant rats from the seventh to the twentieth day of pregnancy resulted in offspring with unchanged body weights, but with significant increase in brain weights when compared with normal. However, our investigations provide no evidence for growth hormone’s action on the intact early postnatal brain. In order to understand more clearly the action of growth hormone on brain development at least two further experiments are necessary. One, to repeat the conditions of Zamenhof’s investigations, and two, to increase the dosages of growth hormone administered to the postnatal rats. References 1. ASLIIYG, C. \I:., and H. R. FRANK. development in rats. 1. Normal Plzys. Antlwopol. 21: 524-544. 2. DIABIOILD. on brain 3. DIAMOND, enriched Newel., 4.
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