The Embryoegnesis of Trypan-blue Induced Spina Bifida Aperta and Short Tail in the Rat

R. G. LENDON

Introduction Previous studies have drawn attention to the fact that various forms of spina bifida

aperta can be induced in rat and other embryos by injection of trypan-blue dye at appro- priate stages of gestation. This work indicates that the probable cause of myelocele lesions is neural dysraphism at an early age.

Lendon (1968) confirmed the earlier observations of Hamburgh (1954) and Beck and Lloyd (1963) that accumulations of fluid (blebs) or haematomata can occur in the trunk region of early-treated embryos, and described some of the histological features of these lesions. The present study, using a large series of rat embryos, is an attempt to discover whether the bleb and haematoma-type lesions are intermediate stages during the embryo- genesis of trypan-blue induced neural dysraphism, or whether these embryos are destined to die and be resorbed.

Material and Method A total of 140 pregnant Wistar-derived rats were injected with a total of 30mg 1 per cent

aqueous trypan-blue i n the manner previously described (Lendon 1968). Mothers were sacrificed at various stages between 10 and 20 days gestational age. !20 malformed embryos from the 12 to 20 day age-group were paraffin-processed and sectioned either transversely or sagittally at IOpm. All the embryos of 10 to 12 days gestational age were sectioned at IOpm in their embryonic membranes (yolk sac and amnion) to avoid damage. The sections were stained with Harris haematoxylin and eosin.

I n total there were 95 litters of 12 to 20-day embryos and 45 litters of 10 to 12-day embryos.

Results (Tnhl:~ I )

differed markedly. Both age-groups provided normal and resorbing embryos. although the proportion\

I2 ro 20-da,. Enihryos Of the older embryos. approximately 40 per cent were superficially normal, more than

one-third were resorbing and 15 per cent had anomalies of the neuraxis of the trunk or tail. Additional anomalies encountered were microphthalmia, truncal oedema, exomphalos. rxencephalus. gross hydrocephalus, partial duplication of the tail, and sirenomelia.

Anomalies of the neuraxis fell into the three categories described previously (Lendon 1968), i.e. groups with a myelocele and normal tail, with a myelocele and absent or short tail. or with only an absent or short tail. The most common type was the myelocele with

Department of Anaton?). University of Manchester, Stopford Building, Oxford Road. Manchester M I 3 9PT

DEVELOPMENTAL MEDICINE AND CHILD NEUROLOGY. 1975, 17. Supp. 35

No. of Age (dn.v.s) Iiriers Nornint

No. (%)

Abnormal A &/if iona! Resorbed anonrolies Toial

M + T * M-T* 1 ST*

No. (%) No. (%)

!?: 15) 13f 125

I I I I

Dysraphia

12 52 (44) 41 (34) 16 (13) 88 (41) 91 (42) 24 ( I 1 ) 26

28 20 97 (44) 82 (37) 25 ( I I ) 9 61 (58) 29 (27) I5 (14)

11 1 (40) 128 (46) 23 (8)

I 1 I

11: 103

No. (%)I No. (:,:,)I No. I No.

21 82 (41) 64 (32) 4 (2) 24 53 (23) 66 (28) 10 (4)

37 (18) I0 (5) 2 (1) 86 (37) 233

Fig. 1 (/<fi). Sagittal section of 154-day embryo showing myelocele, notochord deflection and normal tail. Fig. 2 (right). Transverse section of myelocele at 1st days. Note abnormal chondrification of neural arches.

4

R. G . LENDON

Fig. 3 ( ~ x J w ) . Transverse section of myelocele with slit-like canal. Fig. 4 (rich/). Sagittal section to show dorsal fork of notochord (arrow) at point of ventral deflection of notochord. Insert shows higher power of next serial section.

structure similar to those encountered in human diastematomyelia. Embryos at 131, days with a myelocele often exhibited a haematoma beneath the cephalic

end of their open neural plate (Fig. 5). Older embryos only occasionally showed histological signs of a haematoma beneath their myelocele (Fig. 6).

The two short-tailed categories had the features described previously (Lendon 1968). The notochord either terminated abruptly in the lumbo-sacral region (usually attached to an abnormal spinal cord) or, more frequently, was ventrally deflected and closely opposed to the hindgut or cloaca. Termination or deflection was followed caudally by vertebral agenesis, and by abnormality and termination of the spinal cord, The tail was a thin filament, on average Imm in length. I t was common to find primary malformations of the uro- genital system in embryos with short tails (Lendon and Forbes 1971).

The small number of short-tailed embryos encountered at 12.+ and 139 days had tails which contained small, irregularly-shaped somites and sometimes a small neural remnant. These tails were in the process of regressing, pyknotic nuclei being present in all tissues, and each tail had small blood or fluid-filled spaces at its distal end similar to those described by Hamburgh (1954). In the lumbosacral region, the notochord was ventrally displaced and fused with the developing hindgut.

I0 to I2-day Dnhryo F

There were five main categories of malformation between days 10 and 12 (Table I ) . At 10: days, the most common malformation was increased intercellular fluid or larger

blebs i n the paraxial mesoderm in the thoracic region and below. In many embryos the neural groove appeared to be closing normally in spite of the fluid, but in others the neural plate was dysraphic (Fig. 7). Dysraphia without associated fluid was rare.

Only four of the llf-day embryos had a dysraphic neural plate alone; most exhibited an open neural plate, underlain and everted by blood or fluid (Fig. 8). In these the noto- chord was ventrally displaced and close to, or fused with, the hindgut at a stage when it would normally be attached to a normal neural tube more dorsally. Il+-day embryos with

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DEVELOPMENTAL MEDICINE AND CHILD NEUROLOGY. 1975, 17. SUpp. 35

Fig. 5 (above). Sagittal section of a 13+-day embryo with large haematoma (arrow) beneath myelocele. Fig. 6 (right). Sagittal section to show small haematoma (arrow) beneath myelocele at I54 days.

dysraphia and bleb always looked moribund. Pyknosis was present in several tissues locally or throughout the body.

Discussion The results given in Table I suggest several conclusions. The proportion of normal

embryos apparently does not change from day I I f onwards. This statement, is a generalisa- tion, however, as superficially normal animals which were dead were occasionally found at later stages and were categorised with the resorbing embryos.

The number of resorbingembryos increased after day lo+, although this trend was masked by the small number of litters investigated at 12f and 19t days.

The myelocele undoubtedly results from neural dysraphism and there was no evidence to support secondary opening of a previously closed tube. The two types of lesion described (Figs. 2 and 3) are very similar to two of the lesions seen in human spina bifida aperta (Emery and Lendon 1973). The cord duplications seen in man were not encountered in the rat.

Only 3 per cent of the embryos at days l O i and 1 I + had a dysraphic neural plate on its own, compared with approximately 14 per cent of the older embryos with a myelocele. This suggests that embryos from the other dysraphic categories (with haematoma or bleb) must also contribute to the pool of older embryos with a myelocele. As the I I f-day embryos with a bleb always appeared moribund and would undoubtedly resorb at later stages, it appears probable that 1 l+-day embryos with dysraphia and a haematoma must contribute to the older groups with a myelocele. This is supported by the presence of a haematoma beneath the myelocele in several 124 and 13t-day embryos, and by the occasional haematoma beneath the myelocele at 1st days and later.

An anomaly similar to the dorsal notochordal fork we have described might be responsible for the bony spur that occurs in human diastematomyelia. The most common notochordal defect beneath a myelocele was a minor ventral displacement in embryos with a tail (Fig. I ) . As the vertebral bodies ossify around the notochord, this leads to a minor lordosis of the vertebral row beneath the myelocele. A common finding in human myelomeningocele is vertebral kyphosis, but a small number of rats with spina bifida aperta which we allowed to grow for several months after birth (unpublished work) did not develop k y p h o k I t

6

K. G . I INDON

Fig. 7. Transverse section of 104-day embryo ui th dysraphia and hlebs.

Fig. 8. Transverse section of I I f -day embryo with dysraphia and haematorna. Notochord (arrow) close to hind gut.

\tould appear. therefore. that the rat is not a good model for the study of kyphosis, possibly hecause of differences i n myology between man and the rat. This does not exclude the possibility that a notochordal defect might be a predisposing reason for formation of kyphosis in man.

What causes the notochordal lordosis in the rat? On the 1 Ith day. when the neural tube i s closing. the notochord is attached to the roof of the hindgut ventrally and the neural plate dorsally. I n normal circumstances it would separate from the gut and become asso- ciated with the closed neural tube. Dysraphic 1 14-day embryos mostly have notochords which are not attached to the open neural plate. but are close or still attached to the hindgut (Fig. 8). Therefore either the notochord is prematurely released from the neural plate or fails to separate adequately from the hindgut. The latter suggestion seems the most likely. hecause it can be used to explain a series from complete notochord attachment to the gut. through partial separation. to separatiori with ventral lordosis. Trypan-blue probably causes ;I transient nutritional deficiency in the embryo because of its action on the yolk sac. This i \ overtly manifest by retarded growth (Berry 1970) and leads to a developmental delay xhich. when severe enough, can cause neural non-closure and delayed separation of the notochord from the hindgut. In its most extreme manifestations, it could lead to total lack of separation of the notochord from the gut and retarded tail-bud development. This would cause short-tailed animals and, depending on whether the effect was maximal early or late. these embrbos might or might not have a myelocele. The most severe manifestation is h i renomelia.

Jelinek ef trl. (1971) have demonstrated i n the chick how increasing doses of 6-azaurddine can lead from ;I raised incidence of niyeloschisis alone to an increase in ‘rumplessness’

7

DEVELOPMENTAL MEDICINE AND CHILD NEUROLOGY. 1975, 17. Supp. 35

(caudal regression syndrome), and it is not difficult to fit the findings of this present study with the numerous publications of Jelinek and colleagues on the action of trypan-blue and other teratogens on chick embryos. Jelinek et al. (1970) suggested that short tail might result from compression and damage of the caudal proliferation centre in the tail-bud by blebs or haematomata. If this was happening in the present study, it might explain why the tail starts to grow but later regresses. The anlage for the notochord must be damaged, with a resulting absence of notochord in the rudimentary tail.

Vertebral agenesis in short-tailed animals at levels caudal to the point of notochordal deflection towards the gut is caused by the absence of the inductive effect of the notochord.

The other fact to emerge from Table f is that the large number of embryos with paraxial blebs at 104 days is hardly represented at 114 days. It appears that a large number lose the fluid and appear as normal embryos on the following day. Disappearance of bleb fluid is recorded by Kaplan and Grabowski (1967) after moderate hypoxia of chick embryos. From our results, it seems likely that the haematoma at 1 I+ days forms by coalescence of paraxial blebs into a single fluid mass beneath the neural plate, with subsequent extravasa- tion of whole blood from the dorsal aortae into the bleb. Severely ‘blebby’ embryos die before this stage, although this is not adequately indicated in the results.

These results indicate that some of the l o t d a y embryos with blebs do survive, to be found with a myelocele at later stages. Is the fluid responsible for non-closure or simply an associated phenomenon? Neural non-closure was occasionally found in the absence of fluid, which shows that it can take place on its own. The few early human embryos which have been described with myeloschisis have not exhibited blebs. Blebs may be purely an incidental finding in these experiments and only become significant if they cause damage to the developing tail-bud and thus lead to sacral agenesis and/or short tail.

and Mr. A. Tunstill for taking the photographs. Acknowledgernenfsr I thank Action for the Crippled Child and the Richard Fund for financial assistance,

SUMMARY The study of litters from 140 Wistar-derived rats injected with trypan blue during gesta-

tion leads to the conclusion that a myelocele can result from faulty closure of the neural plate and that this is accompanied in many cases by blebs in the paraxial mesoderm. Haematomata usually underlie the open neural plate at an early stage and they form by extravasation of blood from the dorsal aortae into the blebs.

Local ventral deflection of the notochord beneath a myelocele probably results from delayed separation of the notochord from the hindgut. Complete failure of separation and abnormality induced in the tail-bud could result in sacral agenesis and/or a short tail.

All these malformations may result from the varying severity of the action of the trypan blue at different developmental stages.

RESUME EmbrvogPnPse du spina b$da induit au bleu de trypan et courte taille die; le rat

L’ttude des porttes provenant de 140 rattes de race Wistar ayant requ des injections de bleu de trypan durant la gestation conduit A la conclusion qu’un myCloc6le peut resulter de la fermeture defectueuse de la plaque neurale et qu’il est accompagni dans de nombreux cas par des cavitts dans le mtsoderme para-axial. Des htmatomes sont gtneralement observts sous la plaque neurale au stade precoce; ils se forment par exsudation de sang de l’aorte dorsale dans les cavitCs.

8

R. G. LENDON

La deflection ventrale locale de la notochorde au dessous du myClocele provient probable- ment d’une sepraration retardee de la notochorde et de I’intestin posterieur. Un difaut complet de separation et I’anormalitC resultante dans l’origine de la queue peut provoquer une agCnCsie sacrke etlou une queue courte.

Toutes ces malformations peuvent provenir de la gravite variee de I’action du bleu de trypan i differentes Ctapes du developpement.

ZUSAMMENFASSUNG Die Enihryogeneve vow Trypun-hlau hervorgerufener Spina bijida apertn imrl kurzeni Sch M.atiz

hei der Rntte Die Untersuchung des Wurfs von 140 Wistar-abstammenden Ratten, die wiihrend der

Gestation mit Trypan-blau injiziert wurden, fuhrt zu der Folgerung, dap eine Myelo- meningocele durch einen falschen Schlufl der Neuralplatten entstehen kann und dafl dies i n vielen Fiillen begleitet wird von Blaschen irn paraaxialen Mesoderm. Zu einem friihen Stadium liegen Hamatorne gewohnlich unter der offenen Neuralplatte, und sie entstehen durch ein Blutextravasat der dorsalen Aorta in die Blaschen.

Die ortliche ventrale Deflektion der Embryonalleiste neben einer Myelomeningocele entsteht wahrscheinlich durch eine verziigerte Trennung der Embryonalleiste vom hinteren Intestinaltrakt. Ein volliges Fehlen der Trennung durch eine Abnormitat in der Schwanz- knospe hervorgerufen, kann in einer sakralen Agenesie und/oder i n einem kurzen Schwanz resultieren.

All diese Miflbildungen konnen von der variierenden Schwere der Wirkung des Trypan- blau in unterschiedlichen Stadien der Entwicklung herriihren.

RESUMEN La embriogPnesis de la espina bifidu abiwtrr inducidu con azul-tripan 1’ la cola corta en la

ratu El estudio de camadas de ratas derivadas de 140 Wistar, inyectadas con azul-tripan

durante la gestation, lleva a la conclusi6n de que un mielocele puede resultar de la falta de cierre de la placa neural, y que esto se acompaiia en muchos casos de vejigas en el mesoderm0 paraxial. Generalmente existen hematomas por debajo de l a placa neural abierta en un estadio precoz y se forman por extravasacih de sangre desde la aorta dorsal dentro de las vejigas.

Una deflecci6n local ventral de la notocorda por debajo del mielocele probablemente es el resultado de u n retraso en la separaci6n de la notocorda de su cauce posterior.

Un fall0 completo en la separaci6n y una anormalidad inducida en la cola prodria con- ducir a una agenesia sacra y/o cola corta.

Todas estas malformaciones pueden ser resultado de una gravedad variable en la accibn del azul tripan en 10s diferentes estadios del desarrollo.

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Emery. J . L.. Lendon, R. G. ( 1973) ‘The local cord lesions in neurospinal dysraphism (meningomyelocele).’

Gordon, L. H. , Criqui, M. (1974) ‘Foot deformities in the myelodysplastic newborn rat-with human

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DEVELOPMENTAL MEDICINE A N D CHILD NEUROLOGY. 1975, 17. Supp. 35

Gunberg, D. L. ( 1956) ‘Spina bifida and the Arnold-Chiari malformation in the progeny of trypan blue

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Lendon, R. G. (1968) ‘Studies on the embryogenesis of spina bifida in the rat.’ Developmental Medicine

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