Somite Necrosis and Developmental Malformations Induced by Vitamin a in the Golden Hamster

/. Embryol. exp. Morph., Vol. 13, Part 1, pp. IS, February 1965 Printed in Great Britain

Somite necrosis and developmental malformations induced by vitamin A in the golden hamster

by MIGUEL MARIN-PADILLA and VERGIL H. FERM1 From the Department of Pathology, Dartmouth Medical School

WITH THREE PLATES

INTRODUCTION THE primary teratogenic effects of vitamin A in young embryos, when the vitamin is administered to their pregnant mothers are unknown. The morpho- genesis of experimental cranioschisis and sacral rachischisis, commonly induced by vitamin A, is also still unknown. A variety of methods is now available for the induction of anencephaly (cranioschisis) and sacral spina bifida (sacral rachischisis) in some experimental animals. A method frequently used is the administration of a high dose of vitamin A to the pregnant mother. This has been employed in the rat (Cohlan, 1953; Giroud & Martinet, 1955) and in mice (Kalter & Warkany, 1961). In the present experiments, the golden hamster was used as the experimental animal. This study is divided into two parts. The firstpar t consists in the establishment of a method for the induction of developmental malformations in the golden hamster by vitamin A. This part will include an analysis of the results, the study of the vulnerability of the different embryonic stages to the drug and it will establish the critical period for the induction of a given malformation by this method. The second part consists of the study of the effect of vitamin A upon embryonic tissues when administered on the 8th day of gestation, this day being the critical period for the induction of cranioschisis and sacral rachischisis. Investigations of the teratogenic effects of vitamin A on young hamster embryos and its probable mode of action in causing cranioschisis and sacral rachischisis are the objectives of this paper.

MATERIALS AND METHODS Fifty virgin female golden hamsters, Mesocricetus auratus, were used for the first part of the experiment. The females were placed with the males in the early evening hours and, if observed to be in estrus, they were left together overnight. 1 Authors' address: Department of Pathology, Dartmouth Medical School, Hanover, New Hampshire, U.S.A. 1 2 M. MARIN-PADILLA and V. H. FERM The following day was considered to be the first day of gestation. The impreg- nated females were placed in separate cages. Fifty pregnancies were obtained: thirty-three pregnant hamsters were treated and seven used as controls for the first part of this study. A single dose of 20,000 U.S.P. units of vitamin A was administered by gavage (stomach tube). The vitamin was administered to groups of hamsters (first part of the study) in the morning hours of the 5th, 6th, 7th, 8th, 9th, 10th and 11th day of gestation. All animals were sacrificed on the 14th day of gestation. Gross examination of all embryos was made at that time and the types of malformation as well as the number of malformed fetuses in each group were recorded. From these data, the 8th day of gestation was established as the critical teratogenic period for the induction of cranioschisis (anencephaly) and sacral rachischisis (spina bifida) in the golden hamster. For the second part of this study ten additional pregnant hamsters were used. They all received the same amount of vitamin A (20,000 U.S.P.) by gavage on the 8th day of gestation. The animals of this group were sacrificed 6, 12 and 24 hr. and 4, 5 and 6 days after the administration of vitamin A. Serial sections were made of the gestation sacs of the animals sacrificed on the 8th and 9th days, and of the isolated embryos of later stages. All sections were stained with hematoxylin and eosin. RESULTS First part The first part of this study is summarized in the two tables and the Text-figure. Table 1 shows the number of treated animals with the total number of living, malformed and resorbed fetuses for each day of gestation treated. Table 2 summarizes the number of fetuses affected and the percentages of each malformation obtained by this method. The terms used in this table need some comments. Anencephaly is synonymous with cranioschisis and exencephaly. Anopia includes unilateral or bilateral microphthalmia and anophthalmia. Bulging eyes corresponds to hypoplasia of the orbits with protrusion of grossly normal eyes. Spina bifida, in the majority of cases, refers to an occult sacral rachischisis, only few fetuses showing an open meningomyelocele. Rib defects include fusion, hypoplasia and hyperplasia of one or more ribs. Harelip is commonly associated with cleft palate. Limb defects include gross malformations

TABLE 1 Number of treated pregnant hamsters with the total number of living, abnormal and dead fetuses obtained per treated {vitamin A) day of gestation 5th 6th 7th 8th 9th 10th 11th Controls Mother treated 4 4 3 10 4 5 3 7 Living fetuses 45 53 14 89 40 48 33 71 Abnormal fetuses 0 4 5 58 20 36 0 0 Resorbed fetuses 2 2 23 34 2 14 2 4 Developmental malformations induced by Vitamin A

TABLE 2 Types and percentages of malformations obtained per treated {vitamin A) day of gestation in the golden hamster Anen- Bulging Spina Rib Limb Day cephaly Anopia eye bifida defect Harelip defect Hernia 5th — — — — — 6th — — — — 4(7-5) — 7th 1(7-1) 3(21-4) — 1(7-1) — 3(21-4) — — 8th 14(15-7) 2(2-2) 28 (31-4) 11 (12-3) 33(37-0) 2(2-2) — 2(2-2) 9th — — 18(45-0) — 3(7-5) — — — 10th — — — — 5 (10-4) — 36(75-0) — 11th __ __ of the fingers and toes and anomalous position of the limbs. No phocomelic fetuses were found in these series. Hernia refers to a large umbilical hernia. The sensitivity of hamster embryos to the teratogenic effects of vitamin A shown by fetal mortality and the incidences of malformations on different days of gestation are shown in Text-fig. 1. The sensitivity of the embryos varies greatly with the stage of embryonic development. The early embryonic stages before implantation, including the blastocyst stage (5th day of gestation), appear to be resistant to the dose of vitamin A used. Mild teratogenic effects begin to

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101- :• 5 • 7 4-2 ': 4-8 :• :• 5-3 IT ri 5th 6th 7th 8th 9th 10th 11th Totals Controls TEXT-FIG. 1. Sensitivity of hamster embryos to vitamin A expressed as the percentages of fetal mortality and of fetal malformations. 4 M. MARIN-PADILLA and V. H. FERM appear on the 6th day of gestation which corresponds to the beginning of implantation (Graves, 1945). Minor malformations (rib defects) are induced on that day. The administration of vitamin A on the 7th day of gestation produces the highest embryonic mortality in the present study. Major developmental malformations (anencephaly, cleft palate and cleft lip) are first observed after treatment on that day which corresponds to the beginning of development of the embryonic body. The results obtained after administration of vitamin A on the 8th day of gestation show that the number of major malformations increases and the fetal mortality decreases. The administration of the vitamin on the 9th day produces minor developmental malformations (bulging eyes and rib defects) and a further decrease in embryonic mortality. The 10th day of gestation in the hamster marked another critical period of sensitivity to vitamin A with a new increase in fetal mortality, and frequent malformations of the extremities. On the 1 lth day the hamster embryos are resistant to the teratogenic effect of this amount of vitamin A. No congenital malformations are found in the control hamsters and their resorption rate is considered to be within normal limits.

Second part In the second part of the study all embryos were obtained from mothers which received 20,000 units of vitamin A on the 8th day of gestation. Sections of the embryos, 6 hr. after the administration of vitamin A to the mother, did not show any appreciable histologic change or cellular necrosis. The embryos of this group differ in their developmental stage from late presomite to early somite stages. Twelve hours after the administration of vitamin A to the mother the first appreciable changes are found in the embryos. These changes, found only in the somites, consist of cellular necrosis, represented by rupture of the cells, diffusion of the cytoplasm and the formation of small, round, dense and dark bodies. These bodies may persist in the areas of necrosis and can be found later in older embryos. The changes observed in this group of embryos are minimal, localized and are not present in all the sections of the same embryos. The necrosis is restricted to the somites of the cephalic end of the embryo. The embryos in this group have approximately 7-9 somites and the neural tube has begun to close (Plate 1, Figs. A & B). Twenty-four hours after administration of vitamin A massive cellular necrosis of the somites (Plate 2, Fig. C) is observed in all embryos, the surrounding embryonic tissues being unaffected. There is no necrosis or apparent damage to the neural tissue, the entoderm, or the ectoderm of the embryos (Plate 2, Fig. D). In this group of embryos focal necrosis of the notochord is found for the first time (Plate 2, Fig. D, insert). The embryos of this group belong to a 10-somite or later stage. The neural tube appears to be closed except for a small anterior and a posterior neuropore. It is not possible from the study of these embryos to predict the type of malformation that will later appear. All embryos studied in J. Embryol. cxp. Morph. Vol. 13, Part 1

PLATE 1 FIG. A. Longitudinal section of an embryo (inside uterus) obtained 12 hr. after the administration of vitamin A to its mother. The anterior and posterior neuropores are open. The cephalic somites show focal necrosis. (H. & E. x 25.) FIG. B. Detail of two cervical somites of the embryo depicted in Fig. A, showing focal cellular necrosis (arrows). (H. & E. x 200.)

M. MARIN-PAD1LLA and V. H. FERM (Facing page 4) J. Embryol. cxp. Morph. Vol. 13, Part 1

PLATE 2 FIG. C. Somite of the cephalic region of an embryo obtained 24 hr. after administration of vitamin A to the mother. Maximum necrosis in the somite is observed at this time. (H. & E. x 200.) FIG. D. Gross section of the cephalic region of an embryo (24 hr. after vitamin A administration) showing bilateral somite necrosis. Notice the absence of necrosis or any change in the neural tube. This section is inferior to the anterior neuropore. Focal notochordal necrosis is found in this section and in the inset. (H. & E. x 100; inset x 200.) M. MARIN-PADILLA and V. H. FERM {Facing page 5) Developmental malformations induced by Vitamin A 5 this group are affected by cellular necrosis of the somites; some are more severely and extensively affected than others. The fetuses obtained on the 12th, the 13th or the 14th day of gestation from mothers treated on the 8th day with vitamin A, show the malformations clearly. Fetuses with cranioschisis and exencephaly were serially sectioned for micro- scopic study. There are no embryos in this group with an open sacral meningomyelocele. However, some show the typical bulging sacral area characteristic of an occult sacral rachischisis. These cases were also serially sectioned for micro- scopic study. Gross sections of the head of fetuses with cranioschisis, obtained on the 12th day of gestation, show focal necrosis of the notochord with fragmentation of its capsule. Also, areas of necrosis are found in the superior aspect of the cartilaginous blastema of the basilar portion of the sphenoid bone. This last necrosis, in longitudinal sections of the head, accompanies and surrounds the cephalic end of the notochord. The necrosis of the notochord and of the surrounding tissue is indistinguishable from the necrosis seen in the somites of the early embryos described previously. In a well-established experimental cranioschisis with exencephaly (fetuses obtained on the 14th day of gestation) the cephalic notochord (the portion of notochord anterior to the odontoid process of the second cervical vertebra) is narrower, shorter and less well outlined than in the normal fetus. Plate 3, Figs. I & J, show longitudinal sections of an abnormal skull (cranioschisis) and of a normal one. There is no cellular necrosis in any of the structures of the head in this group of fetuses. In experimental sacral rachischisis a notochordal abnormality is found in all embryos studied on the 14th day of gestation. Portions of the sacral notochord are affected, usually a region two or three segments in length. No changes in the notochord of other regions of the vertebral column are found (Plate 3, Fig. G). The abnormal notochord consists of hyperplastic tissue occupying a large part of the vertebral body. The vertebrae with notochordal changes are morphologically abnormal. The shape (Plate 3, Figs. G & H) of the abnormal notochord varies from case to case, assuming all kinds of anomalous configurations. Occasionally, a centrally placed area of necrosis is found in the notochord. This necrosis is surrounded by what appears to be fragments of necrotic notochordal capsule (Plate 3, Figs. E & F) and because of the central location of this necrotic material it may represent an area of previous notochordal necrosis. These necrotic areas are always surrounded by a hyperplastic notochord. DISCUSSION The 8th day of gestation was selected for special study because it yields the highest percentage of cranioschisis and sacral rachischisis. The first indication of the teratogenic effects of vitamin A are observed in the somites of the cephalic end of the embryos 12 hr. after administration of the vitamin. These changes are minimal and localized to the somites of the cephalic portion of the embryo. At 24 hr. the maximum effect is apparently obtained. The changes at this time are 6 M. MARIN-PADILLA and V. H. FERM more generalized, affecting other somites, but the most severely affected still are the most cephalic in location. At this time (24 hrs. after treatment) the first notochordal changes are observed, consisting of focal necrosis in the cervical and cephalic portions of the notochord. Later, 4 and 6 days after treatment, some embryos show schisis of the skull and exencephaly with areas of degeneration, while others show a bulging of the sacral region. Some cellular debris is found in the sphenoid blastema in embryos 4 days after treatment. Six days after treatment, in induced cranioschisis, the cephalic portion of the notochord is narrower and shorter than normal. In sacral rachischisis, focal necrosis and hyperplastic changes in the notochord are found. The predominant cephalic location of the somite necrosis induced by vitamin A on the 8th day is significant, since these embryos are known to have the highest percentage of cranioschisis. This developmental malformation is considered to be the result of a failure of closure of the anterior portion (anterior neuropore) of the neural tube due to primary somite necrosis. Somite necrosis is considered to cause an abnormality of the axial mesoderm (from abnormal segmental derma- tomes and sclerotomes) which then fails to support and protect the neural tube. The neural tube remains open and exposed and soon begins to degenerate. Those embryos with an open anterior neuropore show somite necrosis only. No changes are noted in the neural tube which is still open in these early stages. The neural tube presumably degenerates thereafter for lack of closure and protection. The occult variety of sacral rachischisis induced by this method shows an hypertrophied neural tube in contact with the skin and subcutaneous tissue. Hyperplasia and focal necrosis of the notochord are found in the sacral regions in these embryos. The associated sacral vertebrae are always severely malformed, in that they lack the posterior neural arches or they may have short and deviated arches which never completely enclose the neural tube. The neural tube, then, lacks protection from the vertebral skeleton in the affected area. The neural tube in these cases appears to be relatively normal, which suggests that it was closed at the time of somite injury. This malformation is considered to be the result of somite necrosis occurring after the closure of the posterior neuropore. The abnormal mesodermal tissue resulting from somite necrosis then fails to give proper support to the neural tube and is responsible for the vertebral abnormalities. All embryos studied 12 and 24 hr. after treatment have shown somite necrosis. The only difference between them is in the number of somites affected and the severity of the necrosis in a given somite. Rarely, the entire somite is destroyed. These differences are due to the developmental stage of the somite and are reflected in the type of malformation induced upon mesodermally derived tissue. Minimal damage to the somites may account for the rib defects. Moderate damage may account for vertebral malformations, e.g. occult sacral rachischisis. Extensive destruction may account for the most severe malformation of the head skeleton in cranioschisis. /. Embryol. e.xp. Morph. Vol. 13, Part 1

PLATE 3 FIGS. E and F. Low- and high-power views of a sacral vertebra in an embryo with an occult sacral rachischisis (6 days after vitamin A administration) showing focal notochordal necrosis and notochordal hyperplasia. Notice in Fig. F the rupture and fragmentation of a probable capsule around the necrotic notochord. (H. & E. x 100 and 200 respectively.) FIGS. G and H. Low- and high-power views of a longitudinal section of the sacral region in an embryo with an occult sacral rachischisis (6 days after treatment) showing the abnormal shape of the notochord and anomalies of the sacral vertebrae. (H. & E. x 100 and x 150 respectively.) FIG. 1. Longitudinal section of the base of the skull in a fetus with cranioschisis showing the course of the cephalic notochord. (H. &E. x 50.) FIG. J. Longitudinal section of the base of the skull in a normal fetus (same age as fetus of Fig. J) showing the cephalic notochord. (H. & E. x 50.) Note; Figures I and J have been marked with India ink to show the course of the cephalic notochord as it was found in subsequent serial sections. M. MARIN-PAD1LLA and V. H. FERM (Facing page 6) Developmental malformations induced by Vitamin A 7 SUMMARY 1. A simple method for the induction of developmental malformation in the golden hamster is presented. The teratogenic agent used consists of a single dose of vitamin A. Correlations between the day of gestation at which the vitamin A is administered and the type and percentage of malformations induced are reported. Young embryos obtained from treated mothers on the 8th day of gestation show somite necrosis within 12 hr. of the administration of vitamin A, reaching its maximum at 24 hr. The somite necrosis is considered to be a basic alteration resulting in an abnormal axial mesoderm which then fails to fulfil its function of lodging and protecting the neural tube. The unprotected and exposed neural tissue secondarily degenerates. Somite necrosis is considered to be the primary defect induced by vitamin A causing developmental malformations such as cranioschisis and sacral rachischisis. When somite necrosis occurs before neural tube closure extensive degeneration of the neural tube follows (e.g. cranioschisis). Somite necrosis occurring after closure of neural tube results in only minimal change in the neural tube (e.g. occult sacral rachischisis). 2. Notochordal alterations (hypertrophy and necrosis) are also found in the young hamster embryo after administration of vitamin A to their mother on the 8th day of gestation. RESUME Necroses des somites et malformations du developpement induites par la vitamine A chez le Hamster dore 1. On presente une mithode simple pour l'induction des malformations du developpement chez le Hamster dore. L'agent teratogene utilise consiste en une seule dose de vitamine A. On signale des correlations entre le jour de la gestation auquel la vitamine A est administree et le type et le pourcentage des malformations induites. De jeunes embryons obtenus de meres traitees le 8e jour de la gestation montrent une necrose somitique dans les 12 heures de Padministration de la vitamine A, atteignant son maximum au bout de 24 heures. La necrose des somites est considered comme etant une alteration fondamentale donnant naissance a un mesoderme axial anormal qui ne remplit pas alors sa fonction de protection du tube neural. Le tissu nerveux non protege et expose degenere secondairement. On considere que la necrose somitique est la deficience primaire induite par la vitamine A, provoquant des malformations du developpement telles que la cranioschisis et la rachischisis sacree. Quand la necrose somitique survient avant la fermeture du tube neural, une degenerescence etendue du tube neural s'ensuit (p.ex. cranioschisis). La necrose somitique intervenant apres la fermeture du tube nerveux n'entraine que des modifications minimes dans ce dernier (rachischisis sacree occulte). 2. On trouve aussi chez les jeunes embryons de Hamster des alterations de la notochorde (hypertrophie et necrose), apres administration de vitamine A a leur mere le 8e jour de la gestation. 8 M. MARIN-PADILLA and V. H. FERM ACKNOWLEDGEMENTS We wish to acknowledge the technical assistance of Miss Marjorie Stearns and Mrs Linda Wilmot. This investigation was supported by USPH grant GM-10210.

REFERENCES COHLAN, S. Q. (1953). Excessive intake of vitamin A as a cause of congenital anomalies in the rat. Science, 111, 535. COHLAN, S. Q. (1954). Congenital anomalies in the rat produced by excessive intake of vitamin A during pregnancy. Pediatrics, 13, 556. GIROUD, A. & MARTINET, M. (1955). Hypervitaminose A et anomalies chez le foetus de rat. Rev. int. vitamin. 26,10. GRAVES, A. P. (1945). Development of the golden hamster Cricetus auratus during the first 9 days. Amer. J. Anat. 11, 219. KALTER, H. G. & WARKANY, T. (1961). Experimental production of congenital malformations in strains of inbred mice by maternal treatment with hypervitaminosis A. Amer. J. Path. 38,1.

{Manuscript received 2nd June 1964)