Paper - On some effects of x-rays on the developing chick embryo (1933)
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Gladstone RJ. On some effects of x-rays on the developing chick embryo. (1933) J Anat. 68: 85-95. PMID 17104466
|X-ray on chicken embryonic development. Note that Entoderm is the historic term for endoderm.
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On Some Effects of X-rays on the Developing Chick Embryo
By R.J. Gladstone University of London, King’s College
and Hector A. Colwell Barnarto Joel Laboratories, Middlesex Hospital, London
In two earlier contributions to this Journal, the present authors in collaboration with C. P. G. Wakeley (6,6) described some of the actions of X-rays upon the development of chick embryos. Those papers dealt with the action of repeated doses, which individually were of the order of those used in ordinary superficial therapy. The present communication has reference to the effect of a single massive dose administered after incubation had proceeded normally for 9 days. In each case four full pastille doses (P.D.) were given.
An ordinary therapeutic induction coil was used: K.v. 120, M.A. 2-5, 20 cm. distance. The eggs were removed from the incubator in boxes lined with cotton-wool, and the very thin layer of cotton-wool covering them was allowed to remain during irradiation.
Dosage was estimated by the Sabouraud pastille, a method which has been shown by Owen and Bowes(16) to give fairly accurate readings within our experimental limits. No account has been taken of screenage due to the eggshell, nor of the effects of the secondary radiations given off by the calcium contained therein. All the eggs were irradiated under the same conditions, and all belonged to the same breed of fowl. It is only fair to recognise, nevertheless, that secondary rays from calcium may play a very important part in biological experiments in which they are concerned. This fact has long been recognised, in connection with the radio-necrosis of bone, and Mottram (15) has shown that it also holds good for soft structures adjacent to bone when both are included in the same field of radiation.
If attempts are made to eliminate this source of complication in experiments, by cutting a window in the shell, we are still faced with difficulties in the production of possible artefacts due to trauma and microbial infection. Goulston and Mottram (14) have shown that error from this source is by no means negligible, and indeed may lead to entirely fallacious conclusions.
The eggs were incubated for 9 days, and were in two series according as filtration of the rays was, or was not, employed.
4p.p. Filter 4 mm. aluminium. Killed at once ” ” ” Killed after 2 hours
” 2 ” ” 4 ” 2 2” ” 2”? 6 2”
» » » ” 24
4p.p. No filter. Killed at once % 2 Killed after 2 hours
ee 2° ”? ”
” ” ” 6 ” ” ° 24 (dead)
After exposure, the eggs which were not to be opened at once, were returned to the incubator for the requisite times. Sagittal sections were made from each embryo in the mid-axial line, and also laterally so as to include the optic vesicle and the genital ridge. All the embryos were found to be alive with the exception of one unscreened specimen in which the egg was opened 24 hours after exposure.
Fixation was effected in a mixture of potassium bichromate, 3 gm., glacial acetic acid, 5 c.c., distilled water, 100 c.c., for 24-48 hours. Specimens were washed in distilled water; transferred to 50 per cent. alcohol; then dehydrated, embedded in paraffin, sectioned and stained with iron-haematoxylin and eosin; van Gieson; or picro-indigo carmine. Details of cell structure were carefully examined throughout the series with a 4 in. immersion lens.
General Changes Produced By Radiation
The changes observed were all destructive in character; the longer the time the embryo was allowed to survive after exposure, the more marked were the changes. With the same dosage the amount of damage was diminished by increased screenage. The general effects were perhaps not so marked as might have been expected: this is probably due to the fact that the longest period of survival (24 hours, screened; 6 hours, unscreened) was insufficient to allow the full effect of radiation to be manifested. The most obvious changes were: a general blurring of the details of cell structure, the cell body appearing granular, and the distinction between cytoreticulum and cytoplasm being ill-defined or completely obscured; defective staining of the cytoplasm with eosin; detachment of epithelium; want of definition and irregularity of cell outline; and a tendency towards fusion of cells and the formation of syncytial masses.
There was frequently thickening and irregularity of the nuclear membrane and a tendency towards pycenosis.
Mitoses were absent in two specimens, each of which had been allowed to survive for a period of 2 hours after exposure to the X-rays. In one of these specimens a 4 mm. aluminium filter was used; the other was unscreened. In specimens killed immediately after exposure, or allowed to survive for 4, 6 or 24 hours, mitoses were abundant; but the individual chromosomes were often irregular in disposition and clumped together in masses.
The red blood corpuscles were often markedly distorted and in some cases fragmented. The capillaries, especially those of the sub-epidermal mesenchyme and central nervous system, were frequently dilated. Those in the subcutaneous tissue were usually full of blood, indicating an incipient stasis: those in the central nervous system were generally empty or contained only very few blood corpuscles.
The embryos were removed entire with portions of the membranes attached. These included parts of the yolk-sac, allantois, amnion and chorion. The effects of radiation were difficult to assess, since these structures normally show both proliferative and degenerative changes: the former might easily be mistaken for hyperplasia or hypertrophy, and the latter for atrophy, resulting from radiation, whereas in fact both conditions occur in perfectly normal specimens.
Fig. 1. Nodule of proliferating cells which projected from the inner surface of the amnion, of a normal chick embryo, incubated 9 days.
In the experimental series, especially in the unscreened series allowed to survive for some time after exposure, the entodermal cells lining the yolk sac were extensively broken down and were irregular in form with large vacuoles. The nuclei, poorly stained, were in many cases liberated by the disruption of the cell bodies and lay free in the cavity of the sac. The mesoderm was less affected than the entoderm, but its fibres were swollen and ill-defined. Many large capillaries and sinusoidal spaces were present; these contained red blood corpuscles in different stages of development and degeneration, many being distorted and fragmented while others were devoid of a nucleus.
The epithelial cells and connective tissue of the yolk sac were in general less deeply stained than the corresponding tissues of the other three membranes. The ectodermal cells lining the amnion were often swollen and their outlines irregular and ill-defined. Localised masses of proliferating cells were occasionally observed, and such masses were also seen in the control specimens (fig. 1).
Cutaneous ectoderm. The superficial epithelial strata are frequently separated from the subjacent mesoderm. This becomes more marked with increasing exposure and diminishing filtration. In some cases there is shedding of the epitrichial layer, while the deeper layer of granular cubical cells remains. In the irradiated specimens the cell outlines are less distinct, the staining less intense and the cytoplasm more granular than in the controls. Mitoses are sometimes present, and in some specimens, allowed to survive for some time after exposure, there is dilatation of the subepidermal capillaries.
Neural ectoderm. One of the most obvious changes is a marked enlargement of the capillaries of the brain and spinal cord. For the most part these are situated at the junction of the ependymal and mantle layers. Since the spaces are almost empty and stand open, they are very conspicuous. They are present in both screened and unscreened specimens, but are most conspicuous in the latter when allowed to survive exposure for 6 hours. The cellular elements of the brain and spinal cord are not so well defined in the irradiated specimens as they are in the controls. Staining was less bright and more diffuse in the experimental series, and with the exception mentioned on p. 86 mitotic figures appeared to be equally frequent in the different categories of the experimental series and in the controls. The chromosomes were however often mis-shapen, irregularly disposed, or clumped together. Nuclei of nondividing cells often showed irregularity of outline and thickening of the nuclear membrane, and there was a general tendency towards pycnosis.
Eye. The general picture is less clear in the irradiated specimens, and the fibres of Miller swollen and irregular. The pigment layer is uneven and folded, the pigment granules irregular and the cell outlines indefinite. The dilated capillaries and large sinusoidal spaces which were observed in the brain and spinal cord were not present in the retina, since the pars optica retinae at this stage is almost avascular. The capillary plexus in the mesenchyme surrounding the optic cup shows marked dilatation, with extravasation of red “blood corpuscles into the extravascular spaces around the retina.
Examination of the epithelial lining of the respiratory and digestive tracts, and of the secretory cells of the liver and pancreas, showed general blurring of cell outlines in the irradiated specimens. In some cases the free borders of the cells were “‘frayed-out”’ with consequent escape of the cell contents into the adjacent lumen.
The cytoplasm generally showed_a granular opacity, and the nuclear membranes were often thickened and irregular in shape. In the liver the endothelium lining the sinuses was not markedly affected.
Thymus. The thymus glands of chick embryos of 8-10 days’ incubation are passing through a transition stage, from avascular and purely epithelial organs to the later condition in which the lobules are invaded by capillary vessels, and a reticular tissue very rich in lymphocytes is developed. The epithelial (entodermal) cells are rounded or polygonal, and the large number of mitoses indicates rapid growth. In those slightly older embryos which were allowed to survive after the exposure (e.g. a specimen 4 P.D., 4 mm. filter, killed 24 hours after exposure) penetration of vessels had advanced to a stage in which they had reached the central parts of the lobules, and a distinction between cortex and medulla was apparent. Thus the exposure to X-rays had not arrested the development and differentiation of the gland. Growth was still active as shown by the frequency of mitoses, and the organ had entered upon a new phase of development. Lymphocytes were not present in the gland itself, although in the blood vessels of the surrounding fibrous capsule there were some small spherical cells with relatively large nuclei. .
Generally speaking the thymus glands of irradiated specimens showed no special features, though the ordinary cell changes—granulation of cytoplasm, thickening of nuclear membrane, etc.—were well marked. This lack of special sensitivity in the thymus of chick embryos at this stage of development is due to the low degree of differentiation attained. The adult thymus containing a reticular tissue with abundance of lymphocytes is exceedingly sensitive to radiations.
Subepidermal mesenchyme. In some specimens, especially those surviving for the longer periods after irradiation, there is some dilatation of capillaries with occasional local extravasations of blood. The separation of small areas of epidermis, previously noted, sometimes corresponded to the regions in which the capillaries are dilated.
Embryonic connective tissue. The cell outlines are ill-defined and the protoplasmic strands between the cells are often torn through and irregularly retracted. Sometimes the endothelial walls of the capillaries are ruptured, and the blood extravasated into the tissue spaces.
Cartilage. Very little change could be detected in the cartilage, especially in those specimens which were killed at once. In some the matrix was more granular and the contrast between the staining of the cartilage cells and the matrix was less evident than in the controls.
Muscle. Skeletal, cardiac and unstriped muscle showed no obvious changes. In some of the specimens allowed to survive after the exposure both muscle fibres and interfibrillar substance had a finely granular appearance.
Blood (figs. 2, 3). In general the red blood corpuscles in the vessels and vascular spaces of the irradiated specimens are more granular and irregular in outline than in the control specimens. The cytoplasm is less deeply stained with eosin and the nucleus less sharply differentiated with regard to its internal structure. A considerable number of corpuscles is distorted and in some cases they are fragmented.
The degree of staining of the nucleus with haematoxylin varies very much, some nuclei being deeply stained, while others are quite pale. Red blood-cells are sometimes seen which do not possess a nucleus and some cells have the appearance of a space from which the nucleus has been extruded, such as is occasionally seen in developing mammalian corpuscles. These are found not only in the irradiated specimens but in normal control specimens at the same stage of development.
Fig. 2. Typical cells present in the blood of a 9-day chick embryo. Control specimen.
The appearance of the mesonephros is very variable. This is largely due to normal degenerative changes in the Wolffian body at this stage of development, in addition to changes produced as a result of the radiations. Consequently in assessing the effect of the radiations, only a general estimation of the differences in the degree of disintegration in the experimental and control specimens could be attempted.
The principal changes observed were: loosening of the epithelium lining the tubules with separation of the individual cells; these being frequently shed off into the lumen of the tubule. Defective staining of the cell bodies with eosin. Vacuolation of the cytoplasm. Irregularity of the free border of the cells, the edges of which were often frayed. Great variability in the appearance of the nucleus, which was sometimes pycnotic, sometimes feebly stained and fragmented. The red blood corpuscles show every stage of degeneration and variation in the degree of staining. Some of the nuclei are pyecnotic, others pale or unstained, and in some corpuscles the nucleus in absent. Haemorrhages into the intertubular spaces are frequent and there is a considerable amount of cell detritus and coagulated fibrinous material. It is probable that many of the distorted and degenerated red blood corpuscles which are found in the general circulatory system at this stage of development in both the normal and experimental series originate in the mesonephros and are swept from this organ by the blood stream into the general circulation, where under normal conditions they will break down still further and become absorbed.
Fig. 3. Typical cells from the blood of a 9}-day chick embryo, exposed to 4 P.p. unfiltered X-rays, and allowed to survive 6 hours after the exposure. Some large oval cells containing refractile granules of varying size are shown. These are seen in the control as well as in the irradiated specimens and are probably detached entodermal cells from the yolk sac, laden with yolk granules undergoing absorption. Many of the erythrocytes are distorted and fragmented.
Genital glands. Although differentiation of the gonads has commenced, it is not far advanced at this stage and changes produced by the action of X-rays are not so pronounced as in the adult ovary or testis. In the ovaries of the irradiated specimens the genital cells were in some cases not quite so clearly defined as in the controls, and the odplasm was more granular. In some the capillary vessels were dilated both in the cortical and medullary zones. There were no obvious changes in either the genital or genitaloid cells of the testes of the irradiated specimens.
This lack of obvious changes in the genital cells of the experimental series is in marked contrast to the results obtained by Dantschakoff and Lacassagne (13). These authors describe complete destruction of the genital cells in both testis and ovary. The difference however may readily be explained when it is realised that these results were obtained by experiments carried out on chick embryos, (1) at much later stages of development, (2) that three separate doses were administered at intervals of 8 days, and (8) that the chicks were allowed to survive after the exposures up to periods of 17-21 days’ total ineubation.
Some of the most important work in this country of the effects of X-rays on mitosis was that of Strangeways (22) who exposed tissue-cultures obtained from the choroid of chick embryos of 6-7 days’ incubation. Soft rays filtered through 2 mm. of cardboard were employed. He found in cultures fixed immediately after varying periods of exposure: (1) after 20 minutes fewer cells in prophase were present; (2) after 30 minutes cells in prophase were scarce; (3) cells in the later phases of mitosis begin to diminish in number after 25 minutes; (4) after 35 minutes only one or two are found; (5) if not exposed to X-rays longer than 30 minutes the cells already in prophase pass through the various stages of mitosis and the cells divide normally; (6) after longer exposures some of the chromosomes show granular changes and after an exposure of 50 minutes or longer some show lag in division both during metaphase and the passage of some of the chromosomes or their fragments to the poles of the spindle.
A second series consisted of cultures which were fixed after 80 minutes’ incubation subsequent to varying periods of exposure. This period was selected by Strangeways because he found it allowed time for cells to pass through the phases of mitosis and complete their division: (1) after exposures of 20 minutes the appearance of dividing cells practically ceases; but (2) if cultures in which mitosis has ceased are returned to the incubator for some hours, cells in mitosis are again found, and unless too heavy a dose of X-rays has been given they become plentiful.
A synopsis of more recent work at the Strangeways Laboratory upon the action of radiations on tissue cultures has been given by Cox and Spear (12). Experiments by Strangeways and Fell (23), upon somewhat similar lines to our present work, show the disappearance of mitotic figures after 80 minutes’ (post-radiation) incubation, and their re-appearance after 24 hours. In our experiments complete disappearance of mitoses was noted 2 hours after a single exposure, and re-appearance in specimens fixed after 4, 6 and 24 hours’ (post-radiation) incubation.
Our observations thus completely agree with the results previously obtained by Canti and Donaldson(@), who, working with radium on tissue cultures from chick embryos, found mitoses present after 1 hour’s exposure, but absent after 2 hours and longer periods. If however the radium was removed after an exposure of 2~3 hours, mitoses gradually returned in the course of a few hours.
In none of their experiments was there any evidence of the radium producing an increase in the number of mitoses.
Spear (18,19, 20, 21) concludes from his own experiments that the pre-mitotic stage is the most vulnerable phase of the nuclear cycle, so far as radiations are concerned.
It will be necessary here to refer to the use of the term “‘embryonic tissue” with regard to its bearing on the physiological factors concerned in tissue radiation. Bergonié and Tribondeau! showed in 1906 that X-rays act with greater intensity on cells which are actively reproducing themselves than those which are dividing less rapidly and also that their action is greater on cells which are preparing for their functional activity than those which are quiescent. In applying this general law to the conditions met with in experimental work on the later stages of growing embryos one must not infer that a developing organ, such as the thymus or testis is more sensitive to the action of X-rays in all stages of its development than it is in the adult. It is a well-known fact that the thymus and the testis of the adult are extremely sensitive to X-rays. The sensitivity of the fully developed thymus, however, is due to the presence of a large number of lymphocytes, these being the specially sensitive elements in its constitution. In the early stages of development they are not present, consequently there is no special sensitivity of the organ at this stage. Moreover in the testes where spermatogenesis continues throughout adult life both factors enunciated in the law are present and the adult organ is accordingly particularly sensitive to radiations, whereas during that period of its development which follows the initial stage of proliferation in which the cells are in an undifferentiated, quiescent condition pending the active growth and differentiation which will take place later at the attainment of sexual maturity very little change is produced by exposure to X-rays. A more highly differentiated and functional adult organ may thus be actually more susceptible to X-rays than the less differentiated developing organ, and the small amount of change which is produced by the radiations on the embryonic gland confirms the validity of the law rather than refutes it. The term “embryonic tissue” as employed in the description of certain tumours is usually applied to a tissue composed of rapidly proliferating cells, of large size and rounded form, such as are seen in a morula, and it is this type of tissue which is referred to as “embryonic” in the first condition which is associated with special sensitivity to X-rays. The important second condition, namely the modification which the cells undergo, “dans la préparation des transformations fonctionelles,” appears to be less generally known and apt to be forgotten in comparing the sensitivity of adult with embryonic organs. Thus the susceptibility of a tissue often appears to depend as much upon the number of cells in it which are dividing at any given time as upon any special sensitivity of the tissue or organ itself. The tissues of an embryo at any particular phase of its development vary greatly in the rapidity of their growth in different parts of the embryo. It may be inferred therefore that all embryonic tissue is not equally sensitive. Further, because a tissue is embryonic it is not necessarily more sensitive than the same tissue in the adult.
1 Vide, Regaud et Lacassagne.17).
Although the central nervous system of the adult is generally regarded as particularly insensitive to y- and X-rays, this must not be accepted without reservation. Indeed, although Morowoka and Mott in epitomising their work on the action of y-rays on the central nervous system, state that “‘no gross nervous symptoms' were reported ”’ in the experimental animals, they nevertheless describe and figure very definite structural changes as the result of exposure.
That the young and developing central nervous system with its abundant mitoses should be much more sensitive than that of the adult is only what would be expected.
Although research by the methods of tissue culture occupies a deservedly prominent place at the present time, yet we think that experiments of the type we have described have considerable value. The tissues are irradiated as parts of a living organism existing under natural conditions, with an established circulatory system, and growing in interdependence with each other. The two methods are complementary the one to the other, and either method may afford confirmatory evidence of results obtained by the other.
The action of X-rays on the living tissues of chick embryos, under the conditions of radiation employed in this series, appears to be purely destructive.
The full effect of the rays is not manifested immediately.
The effects of the rays were more pronounced in the specimens exposed to unfiltered rays than in the screened series.
The structures chiefly affected are:
- The cutaneous ectoderm and the vessels of the subepidermal tissue.
- The central nervous system and the epithelium of the sense organs.
- The genito-urinary system.
- The vascular system; this shows dilatation of capillary vessels, and a damaged condition of the endothelium and red blood corpuscles.
Embryonic tissues and organs in which differentiation is more advanced appear to be more susceptible to the action of X-rays than those which are less highly differentiated.
The cellular changes consisted chiefly of a thickening of the nuclear membrane, which was sometimes accompanied by pycnosis. A ground-glass appearance and vacuolation of the cytoplasm, with diminished capacity for staining with eosin. Irregularity and blurring of cell outlines, with a tendency towards fusion of cells.
1 The italics are ours.
We were unable to detect any difference in the number of mitotic figures in the nuclei of the control specimens and that in specimens killed immediately after exposure to X-rays 4 P.D.
Of those allowed to survive for a period after exposure, no mitotic figures were present in specimens incubated for 2 hours subsequent to the exposure.
In specimens allowed to survive 4 hours, mitotic figures were abundant, and in those which were allowed to survive 6 or 24 hours the number present was approximately equal to that in the controls, but there was irregularity in the disposition and form of the chromosomes.
No appreciable difference in the number of mitotic figures was apparent between the specimens exposed to filtered and unfiltered rays.
It appears that a single exposure’ to X-rays 4 P.D. produces a temporary inhibition of mitosis, but after 2 hours’ incubation subsequent to the exposure, a recovery of the power of the cells to divide takes place, similar to that which has been previously demonstrated by Strangeways, Canti and Spear in both chick embryos and tissue-cultures.
Attention was drawn to the danger of mistaking normal processes of proliferation of cells and degeneration of tissue elements which occur in the ovular membranes, gonads and mesonephros of chick embryos about the 9th day of incubation for the effects of X-rays or radium, and the liability to error which exists in observations made upon the chorio-allantoic membrane.
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